Chemical  Pathology 


BEING  A  DISCUSSION  OF  GENERAL  PATH- 
OLOGY FROM  THE  STANDPOINT  OF 
THE    CHEMICAL     PROCESSES     INVOLVED 


BY 

H.  GIDEON  WELLS,  Ph.D.,  M.D. 

'I 

PROFESSOR   OF   PATHOLOGY  IN   THE    UNIVERSITY   OF   CHICAGO   AND  IN 

RUSH    MEDICAL   COLLEGE,    CHICAGO;    DIRECTOR    OI'  THE 

OTHO    S.    A.    SPRAGUB    MEMORIAL    INSTITUTE 


FOURTH  EDITION.  REVISED  AXD  RESET 


PHILADELPHIA   AND   LOXDOX 

W.    B.    SAUNDERS    COMPANY 

1920 


Copyright,  1907,  by  W.  B.  Saunders  Company.    Revised,  entirely  reset,  reprinted  and 

recopyrighted  February,  1914.     Revised,  entirely  reset,  reprinted  and  recopy- 

righted  January,  1918.     Revised,  entirely  reset,  reprinted  and 

recopyrighted  July,  1920. 


Copyright,  1920,  by  W.  B.  Saunders  Company 


PRINTED    IN    AMERICA 


TO 
^  u  5  V  1 3    3^  e  K  1 0  e  n 

THIS   BOOK  IS  RESPECTFULLY  DEDICATED,  AS  A 

SLIGHT  TOKEN  OF  THE  GRATITUDE  AND 

ESTEEM  OF  HIS  PUPIL 


720791 


rUKFACE  K)   IHK  FOURTH  EDITION 


TnK  rapid  growth  of  intorost  in  the  ohomical  problems  of  iiiodical 
and  Ijiological  science  is  shown  by  the  groat  incrcas(>  in  the  amount  of 
mat(M'ial  which  must  be  inchided  in  (>aclisncceedinKe(Ution.  Ahhoush 
this  hit(>st  edition  has  hvcu  subj(>cted  to  extensive  revision  and  many 
miiioi-  alterations,  yet  tlu^  fieneral  i)lan  has  not  been  changed.  The 
rapidly  growing  iiifoiination  concerning  the  nutritional  factoi-s  that 
are  essential  to  growth  and  rej)aii-,  and  without  which  serious  "Defi- 
ciency Diseases"  may  arise,  has  necessitated  the  introduction  of  a  new" 
chapter  to  cover  this  subject,  the  importance  of  which  has  been  ac- 
centuated by  the  war  and  its  sequels.  The  growing  bulk  of  material 
on  the  Reactions  of  Immunity  reciuired  a  rearrangement  of  this 
material,  so  that  a  separate  chajiter  on  Anaphylaxis  and  Allergy  has 
l)een  provided,  foi-  j)ur):)oses  of  convenience.  Numerous  sections 
have  been  entirely  rewritten,  and  few  pages  have  not  required  re- 
vision or  addition. 

In  order  to  prevent  the  increasing  material  that  must  be  included 
from  resulting  in  too  cumbersome  a  volume,  much  more  of  the  matter 
is  printed  in  smaller  type.  It  is  hoped  that  this  arrangement  will 
achieve  its  aim  without  serious  reduction  in  facility  of  use.  The  author 
recognizes  fully  that  it  w^ould  be  easily  possible  to  report  the  existing 
state  of  knowledge  on  the  topics  covered  in  "Chemical  Pathology" 
in  a  much  ])riefer  space,  if  only  completely  established  evidence  weie 
included.  "With  the  object  of  serving  as  a  guide  to  investigators,  and 
with  the  hope  of  stimulating  further  investigations,  much  more  than 
this  minimal  amount  of  existing  evidence  is  included.  It  is  also  recog- 
nized that  the  brief  discussion  of  the  elementary  principles  of  physical 
chemistry  and  the  fundamentals  of  the  physics  and  chemistry  of 
living  cells,  which  constitutes  the  introductory  chapter,  maj^  be  out  of 
place  in  a  work  on  Pathology,  and  the  elimination  of  this  chapter  has 
been  seriously  considered.  Repeated  assurances  of  the  usefulness  of 
such  a  presentation,  however,  have  resulted  in  its  retention,  at  least 
for  the  present. 

As  with  all  previous  editions,  mj'  indebtedness  must  be  acknowl- 
edged to  numerous  colleagues  who  have  kindly  read  over  the  sections 
of  this  book  which  most  closely  concern  their  own  fields,  and  especially 
to  the  members  of  my  Department  and  of  the  Sprague  Institute  who 
have  made  manj^  useful  suggestions.  The  chapter  on  Diabetes  is,  as 
before,  contributed  by  Dr.  R.  T.  AVoodyatt,  Director  of  the  Laboratory 
of  Clinical  Research  of  the  Otho  S.  A.  Sprague  ^lemorial  Institute. 

H.  G.  W. 
Chicago,  III.. 
July,  1920. 


PREFACE  TO  THE  FIRST  EDITION 


During  the  past  score  of  years  the  subject  of  biological  chemistry 
has  attracted  the  attention  and  labors  of  a  constantly  increasing  num- 
ber of  investigators,  many  of  whom  have,  for  one  reason  or  another, 
been  interested  in  pathological  conditions.  Sometimes  the  physiolo- 
gist has  sought  for  light  on  his  problems  in  the  evidence  afforded  by  re- 
lated pathological  conditions.  Frequently  chnicians  have  studied  the 
metabohc  changes  and  the  composition  of  the  products  of  disease  pro- 
cesses. Relatively  seldom,  unfortunately,  has  the  pathologist  at- 
tacked his  problems  by  chemical  methods.  From  the  above  and  other 
sources  have  come  scattered  fragments  of  information  concerning  the 
chemical  changes  that  occur  in  pathological  phenomena.  Only  when 
bearing  upon  conditions  such  as  gout  and  diabetes,  which  concern 
ahke  the  physiologist,  the  clinician,  and  the  pathologist,  have  the 
fragments  been  moulded  together  into  a  homogeneous  whole.  For  the 
most  part  they  still  remain  isolated,  uncorrelated,  frequently  uncon- 
firmed items  of  information,  scattered  through  medical,  chemical, 
physiological,  and  physical  literature. 

It  has  been  the  aim  of  the  writer  to  collect  these  scattered  fragments 
as  completely  as  possible,  and  to  use  them  as  a  basis  for  a  consideration 
of  General  Pathology  from  the  standpoint  of  the  chemical  processes 
which  occur  in  pathological  conditions.  Owing  to  the  diffusely  scat- 
tered conditions  of  the  literature  on  which  this  work  is  based,  it  cannot 
be  claimed  that  all  of  the  many  contributions  from  which  useful  in- 
formation might  be  obtained  have  been  noticed;  but  it  is  hoped  that 
a  sufficiently  thorough  collection  of  material  has  been  made  to  afford 
a  fair  basis  for  a  consideration  of  "Chemical  Pathology."  The  time 
seems  ripe  for  an  effort  of  tliis  nature.  Within  the  past  few  years 
great  and  encouraging  advances  have  been  made  in  biological  chem- 
istry, which  in  many  instances  seem  to  throw  hght  upon  pathological 
processes.  In  medicine,  the  use  of  chemical  methods  in  the  study  of 
clinical  manifestations  has  become  more  general,  and  has  yielded 
valuable  information.  Pathologists  have  come  to  feel  that  the  op- 
portunities for  the  acquirement  of  knowledge  by  means  of  morphologi- 
cal studies  have  become  reduced  to  a  minimum,  while  the  fields  of 
pathological  physiology  and  chemistry  lie  still  almost  unexplored. 
The  development  of  research  upon  the  subject  of  natural  and  acquired 
immunity  has  presented  innumerable  problems,  all  of  which  are 
essentially  chemical.     And  perhaps  most  important  of  all  is  the  general 


8  PREFACE   TO   THE  FIRST  EDITION 

awakening  of  an  appreciation  of  the  importance  of  physiological  chem- 
istry to  mecHcal  science,  which  has  led  to  the  introduction  of  laboratory 
courses  on  this  subject  in  every  medical  school  worthy  of  the  name. 

A  book  on  (^hemical  Pathology  should,  therefore,  seek  to  supply 
information  to  a  varied  group  of  readers.  It  should  furnish  collateral 
reading  to  the  student  who  for  the  first  time  goes  over  the  subject  of 
General  Pathology,  which  his  text-books  usually  consider  chief!}'  from 
the  morphological  standpoint.  It  should  e.xploit  to  the  graduate  in 
medicine  the  advances  that  are  being  made  along  lines  that  are  of 
fundamental  importance  to  clinical  medicine.  It  should  serve  for  the 
investigator  in  biological  chemistry  or  in  pathology  as  a  source  of 
information  concerning  the  ground  upon  which  the  two  subjects  over- 
lap— the  "Grenzgebiete"  of  Patholog}'  and  Physiological  Chemistry. 
And,  above  all,  it  should  afford  a  guide  to  the  sources  of  our  knowledge 
of  these  subjects,  since  nothing  but  direct  familiarity  with  the  original 
reports  of  the  investigators  themselves  can  give  the  student  an  im- 
personal view  of  the  actual  status  of  the  questions  under  consideration. 
On  account  of  this  multiplicity  of  the  objects  in  view,  it  has  often  been 
necessary  to  consider  certain  topics  from  more  than  one  standpoint; 
which  explains,  perhaps,  certain  apparent  irregularities  in  the  style 
and  manner  of  treatment. 

It  has  been  assumed  that  the  reader  has  at  least  an  elementary 
knowlefige  of  organic  and  physiological  chemistry.  For  the  benefit 
of  those  whose  studies  in  these  subjects  date  back  some  years,  it  has 
seemed  advisable  to  include  in  an  introductory  chapter  an  epitome  of 
the  more  modem  views  concei-ning  the  chemistry  of  the  protein  mole- 
cule, the  composition  of  the  animal  cell,  and  the  principles  of  physical 
chemistry,  in  as  far  as  they  apply  to  biological  problems.  The  general 
consideration  of  "Enzymes"  in  Chapter  II  is  written  with  a  similar 
object.  In  discussing  these  fundamental  topics  it  has  seemed  advis- 
able to  omit  detailed  references  to  the  numerous  original  sources, — 
these  may  be  found  cjuoted  in  the  special  text-books  cited  in  the  foot- 
notes; but  in  presenting  the  more  distinctly  pathological  topics  the 
attempt  has  been  made  to  render  all  the  important  literature  available 
to  the  reader  and  investigator.  To  economize  space,  a  complete  bibli- 
ography has  not  been  inserted  when  this  exists  already  collected  in 
some  readily  accessible  review  or  original  article;  hence  the  references 
cited  in  the  foot-notes  will  generally  be  found  to  include  only  the  more 
recent  jHiblications.  These  references  have  ])een  so  selected,  however, 
that  they  will  be  found  to  furnish  bibliographical  matter  sufficient  to 
lead  the  investigator  to  all  the  imj)ortant  literature  on  the  topics 
covered  in  this  book.  As  to  those  subjects  (such  as  gout ,  diabetes,  and 
gast  ro-int(>stinal  i)utrefaction)  which,  .because  of  their  great  practical 
j'linical  interest,  have  already  been  discussed  in  available  monographs 
at  greater  length  than  the  scope  of  this  work  would  permit,  it  has 
seemed  appropriate  merely  to  suiniiiai-iz(^  the  most   recent  views  and 


PREFACE   TO    THE  FIRST  EDITION  9 

julvances,  rclVniiifi;  tlic  leader  to  llic  special  ti'enfise.s  for  llie  jj;eiieral 
and  historical  discussion.s. 

It  is  with  the  greatest  pleasure  tluit  I  acknowledge  my  indeljtedness 
to  manj^  colleagues  in  the  University  of  Chicago,  who  have  kindly  read 
the  sections  of  my  manuscript  that  touch  upon  theii-own  special  fields, 
and  whose  criticism  and  advice  have  been  of  the  greatest  assistance; 
their  mimb(>r  alone  prevents  my  thanking  them  by  name.  Most  par- 
ticularl}',  however,  must  I  express  my  debt  to  my  former  instructor, 
Professor  Lafayette  B.  Mendel,  of  Yale  University,  whose  kindly 
criticism  and  suggestions  have  been  of  inestimable  value.  For  con- 
stant assistance  in  the  preparation  of  the  manuscript,  and  for  the 
revision  of  the  bibliography',  I  am  ind(>bted  to  mv  wife. 

H.  G.  W. 


CONTENTS 


CHAPTER  I 

Paoe 

Introduction' 17 

The  Chemistry  and  Physics  of  the  Cell 17 

Chemistrj^  of  the  Essential  Cell  Constituents 18 

Proteins 19 

Fats  and  Lipoids  (Lipins) 22 

Carbohydrates 23 

Inorganic  Substances 24 

The  Physical  Chemistry  of  the  Cell  and  Its  Constituents 24 

Crystalloids  and  Their  Properties 24 

Colloids 31 

The  Structure  of  the  Cell  in  Relation  to  Its  Chemistry  and  Physics ...  39 

The  Nucleus 40 

The  Cytoplasm 42 

The  Cell-wall 45 

CHAPTER  II 

Enzymes 48 

The  Nature  of  Enzymes  and  Their  Actions 49 

The  Principles  of  Enzyme  Action 50 

The  Toxicity  of  Enzymes 55 

Anti-enzymes 57 

The  Intracellular  Enzymes 62 

Oxidizing  Enzymes 63 

Lipase 70 

Amylase  or  Diastase 73 

CHAPTER  III 

Enzymes  (Continued) .-    •    ■  75 

Intracellular   Proteases    (Proteolytic   Enzymes),  Including  a  Considera- 
tion of  Autolysis 75 

Autolysis 76 

Relation    of    Autolysis    to    MetaboUsm 81 

Defense  of  the  Cells  Against  Their  Autolytic  Enzymes 82 

Autolysis  in  Pathological  Processes '.  85 

CHAPTER  IV 

The    Chemistry    of    Bacteria    .\nd    Their    Products 101 

Structure  and  Physical  Properties 101 

Chemical  Composition 103 

Bacterial  Enzymes 109 

Poisonous  Bacterial  Products 115 

Ptomains 116 

Toxins 120 

Endotoxins 124 

Poisonous  Bacterial  Proteins 125 

Bacterial  Pigments 126 

11 


12  CONTENTS 

CHAPTER  V 

Page 

Chemistry  of  the  Axoial  Parasites 128 

Protozoa 129 

Cestodes 131 

Nematodes 134 

CHAPTER  VI 

Phytotoxi.vs  and  Zootoxixs 13S 

Phj'totoxins 138 

Zootoxins 141 

Snake  Venoms 141 

Scorpion  Poison 150 

Spider  Poison 152 

Centipedes 153 

Bee  Poison 153 

Poisons  of  Dermal  Glands  of  Toads  and  Salamanders 154 

Poisonous  Fish 156 

Eel  Serum 158 

CHAPTER  VII 

Chemistry  of  the  Immunity  Reactions — Antigens,  Specificity,  Anti- 
toxins, Agglutinins,  Precipitins,  Opsonins,  and  Related  Sub- 
jects       159 

Antigens 160 

Xon-Protein  Antigens 161 

Specificity  of  Immune  Reactions 165 

Toxins  and  Antitoxins 172 

Chemical  Nature  of  Antitoxins 175 

Agglutinins  and  Agglutination 178 

Precipitins 184 

Opsonins 188 

The  Meiostagmin  Reaction 1S9 

The  Epiphanin  Reaction 190 

CHAPTER  VIII 

Chemistry    of  the   Immunity  Reactions   (Continued) — Anaphyl.yxis   or 

Allergy,  Abderhalden  Reaction 191 

Anaphylaxis  or  Allergy      191 

Anaphylactogens 191 

Anaphylatoxin 194 

Anaphvlactin 201 

Hay  Fever " 203 

Abderhalden  Reaction 204 


CHAPTER  IX 

Chemistrv    of    thk    Immunity    Reactions     {Continued) — BACTKKiuLV.-iis, 

Hemolysis,  Complement  Fixation,  and  Serum  Cytotoxins  .    .  206 

Serum  Bacteriolysis 206 

Cytotoxins    .    .  " 209 

Hemolysis  or  Erythrocytolysis 210 

Hemolysis  hy  Known  Cheinicnl  iiiid  Pliysical  Agencn-s  211 

Hemolysis  hy  Serum 214 

Hcmolvsis  t.v  liaclcriji 219 


CONTENTS  13 

Paoe 

Hemolysis  by  Vegetable  Poisons 220 

Hemolysis  by  Venoms 223 

Hemolysis  in  Disease 224 

Complement  Fixation  and  Wassermann  Reactions 229 

Cytolysis  in  General 233 

CHAPTER  X 

Chkmical  Means  of  Defensk  Against  Non-Antigenic  Poisons      ....   237 

Inorganc  Poisons 240 

Organic  Poisons      241 

CHAPTER  XI 

I.NFLAMMATION 247 

Ameboid  Motion  and  Phagocytosis 248 

C'hemotaxis      248 

Chemotaxis  of  Leucocytes 250 

Phagocytosis 255 

Theories  of  Chemotaxis  and  Phagocytosis 259 

Artificial  Imitations  of  Ameboid  Movement 260 

Relation  of  the  Above  Experiments  to  the  Phenomena  Exhibited  by  Leu- 
cocytes in  Inflammation 263 

Suppuration 267 

Composition  of  Pus 269 

Sputum 273 

CHAPTER  XII 

The    Chemistry    of    Growth    and    Repair 276 

Proliferation  and  Regeneration 276 

Chemical  Basis  of  Growth  and  Repair 279 

Vitamines  or  Food  Hormones  and  Deficiencv  Diseases 280 

Beriberi •    •    •  ' 283 

Keratomalacia  or  Xerophthalmia 284 

Nutritional  Dropsy  (War  Dropsy  or  Famine  Edema) 285 

Scurvy      286 

Pellagra        287 

Rickets 288 

CHAPTER  XIII 

Distvrbance.s  of  Circulation  and  Diseases  of  the  Blood      290 

The  Composition  of  the  Blood 290 

Hemorrhage 293 

Hemophilia      298 

Anemia  and  the  Specific  Anemias 301 

Secondary  Anemias        301 

Chlorosis 303 

Pernicious  Anemia 305 

Leukemia 309 

Hyperemia 312 

Active  Hyperemia 312 

Passive  Hyperemia 313 

Thrombosis      315 

Fibrin  Formation 315 

The  Formation  of  Thrombi 323 

Embolism 325 

Infarction 327 


14  CONTENTS 

CHAPTER  XIV 

Page 

Edema      330 

Formation  of  Lymph 331 

Absorption  of  Lymph 338 

The  Pathogenesis  of  Edema 340 

Special  Causes  of  Edema 348 

Composition  of  Edematous  Fluids 352 

Varieties  of  Edematous  Fluids 358 

Chemistry  of  Pneumothorax 365 

CHAPTER  XV 

Retrogressive  Changes  (Necrosis,  Gangrene,  Rigor  Mortis,  Parenchy- 
matous Degeneration)       367 

Necrosis 307 

Causes  of  Necrosis      371 

Varieties  of  Necrosis 382 

Fat  Necrosis 387 

Gangrene 391 

Rigor  Mortis 392 

Atrophy 395 

Cloudy  SwelUng      396 

CHAPTER  XVI 

Retrogressive  Processes  {Continued),  Fatty.  Amyloid,  Hyaline,  Colloid, 

AND  Glycogenic  Infiltration  and  Degeneration 400 

Fatty  Metamorphosis 400 

Physiological  Formation  of  Fat    .    *. 401 

Pathological  Fat  Accumulation 401 

Pathogenesis  of  Fatty  Metamorphosis 409 

Processes  Related  to  Fatty  Metamorphosis 412 

Adipocere 412 

Lipemia 415 

Pathological  Occurrence  of  Fatty  Acids 418 

Pathological  Occurrence  of  Cholesterol 419 

Amyloid  Degeneration 421 

The  Origin  of  Amyloid 425 

Hyaline  Degeneration 427 

Colloid  Degeneration 429 

Mucoid  Degeneration 430 

Glycogen  in  Pathological  Processes 432 

Physiological  Occurrence 433 

Pathological  Occurrence 434 

CHAPTER  XVII 

Calcification,  Concretions,  and  Incrustations 439 

Calcification .  439 

Occurrence  of  Pathological  Calcification     ....                                .  442 

Chemistry  of  the  Process  of  Calcification .  443 

Osteomalacia .  447 

Rickets 449 

Concretions 452 

Hillary  Calculi  ,453 

Urinary  Calculi .  459 

Corpora  Aiiiylacea ,  464 

Other  Less  Common  Concretions     .    .                                                     .  465 

Pneumonokoniosis      469 


CONTENTS  15 

CHAPTER  XVIII 

Paob 

Pathological  Pigmentation     471 

Melanin 471 

Lipochroine      478 

Blood  Pigments 481 

Icterus      488 

UrobiUn 495 

CHAPTER  XIX 

The  Chemistry  of  Tumors 497 

A.  Chemistry  of  Tumors  in  General 499 

B.  Chemistry  of  Certain  Specific  Tumors 516 

(1)  Benign  Tumors 516 

(2)  Malignant  Tumors 522 

Multiple  Myelomas  and  Myelopathic  "Albumosuria" 525 

CHAPTER  XX 

Pathological  Conditions  Due  to,  or  Associated  with,  Abnormalities 

IN  Metabolism,  Including  Autointoxication 530 

Uremia .  532 

Toxemias  of  Pregnancy • 540 

Eclampsia 541 

Acute  Yellow  Atrophy  of  the  Liver 547 

Chemical  Changes  of  Acute  Yellow  Atrophy 550 

Acid  Intoxication  and  Acetonuria 555 

Diabetic  Coma 558 

Acidosis  and  Acetonuria  in  Conditions  Other  than  Diabetes  ...  563 

Alkalosis 566 

Acetonuria  Without  Marked  Acidosis 567 

Fatigue 569 

The  Poisons  Produced  in  Superficial  Bums 571 

CHAPTER  XXI 

G  astro-Intestinal  "Autointoxication"  and  Related  Metabolic  Disturb- 
ances      574 

I.  The  Constituents  of  the  Digestive  Fluids      575 

II.  Products  of  Normal  Digestion 575 

III.  Products  of  Putrefaction  and  Fermentation 578 

A.  Protein  Putrefaction 578 

The  Pressor  Bases 584 

Alkaptonuria 586 

Cystine  and  Cystinuria 590 

B.  Products  of  Fermentation  of  Carbohydrates 592 

C.  Products  of  the  Decomposition  of  Fats 592 

Results  of  Gastro-intestinal  Intoxication 593 

Acute  Intestinal  Obstruction 596 

CHAPTER  XXII 

Chemical  Pathology  of  the  Ductless  Glands 597 

Diseases  of  the  Thyroid 597 

The  Functions  of  the  Thyroid      597 

Chemistry  of  the  Thyroid 599 

Chemistrj^  of  Goiter 604 

Myxedema  and  Cretinism 607 


16  CONTENTS 

Page 

Exophthalmic  Goiter 609 

The  Parathyroids 613 

The  Adrenals  and  Addison's  Disease 615 

Addison's  Disease 620 

The  Hypophysis  and  Acromegaly 621 

Thymus  and  Other  Ductless  Glands   . 624 

CHAPTER  XXII 1 

Uric-Acid  Metabolism  and  Gout 626 

The  Chemistry  of  Uric  Acid 626 

Formation  of  Uric  Acid 628 

Destruction  of  Uric  Acid 632 

The  Occurrence  of  Uric  Acid  in  the  Blood,  Tissues  and  Urine 634 

Gout 636 

Uric-acid  Infarcts 640 

CHAPTER  XXIV 

Diabetes 642 

Carbohydrate  Physiology 644 

The  Blood  Sugar 649 

The  State  of  the  Sugar  in  the  Blood 650 

Diose      652 

Trioses       653 

Tetroses 656 

Pentoses 656 

Chronic    Pentosuria 657 

Hexoses      657 

Galactose       661 

Levulose  (Fructose) 662 

Polysaccharides 663 

Glycosurias 664 

Phlorhizin  Diabetes 666 

Pancreas  Diabetes  and  Diabetes  Mellitus 671 


Index 679 


CHEMICAL    PAIIIOLOUY 

CHAPTER  I 

INTRODUCTION 

THE  CHEMISTRY  AND   PHYSICS   OF  THE   CELL 

Since  Virchow  founded  modern  pathology  the  unit  of  all  anatom- 
ical considerations  of  disease  has  been  the  cell,  and  in  physiology  the 
same  unit  has  been  found  equally  useful.  When  either  physiological 
or  pathological  processes  are  studied  from  a  chemical  standpoint,  the 
cell  is  still  found  occupying  nearly  as  fundamental  a  position,  for  ue 
can  seldom  go  back  to  molecules  and  atoms  in  investigating  biological 
problems.  Although  we  know  that  within  each  cell  are  many 
different  chemical  substances,  and  that  numerous  different  enzymes 
and  other  agencies  are  exerting  their  influences  upon  them,  yet  we 
find  that  the  reactions  are  all  profoundly  affected  by  the  environment 
in  which  they  occur,  and  it  is  the  structure  of  the  cell  that  determines 
the  environment  of  its  chemical  constituents.  All  chemical  reactions 
are  modified  by  physical  influences,  and  an  enzyme  may  have  quite  a 
different  effect  upon  a  substance  when  it  acts  in  a  test-tube  from  what 
it  will  have  when  in  a  living  cell,  whose  structure  permits  the  diffusion 
of  one  substance  while  preventing  that  of  another,  and  where  countless 
other  substances  and  enzymes  may  participate  in  the  changes.  The 
cell  is  the  structural  unit  of  the  living  organism,  and  as  l\v  its  physical 
properties  it  modifies  chemical  processes,  so  it  becomes  practically  the 
unit  in  physiological  and  pathological  chemistry.  All  consideration 
of  the  chemistry  of  disease  must  thus  refer  back  to  the  chemistry  and 
physics  of  the  normal  cell,  and  on  this  account  a  brief  resume  of  these 
subjects  may  serve  as  a  fitting  introduction  to  the  strictly  pathological 
matters  to  follow.^ 

As  applied  to  the  animal  tissues,  the  term  "cell"  is  entirelj'  a  mis- 
nomer, for  it  describes  accurately  only  such  forms  of  "cells"  as  are 

'  Of  necessity,  only  so  much  of  the  very  extensive  literature  on  cell  structure 
and  cell  chemistry  can  be  considered  as  will  have  direct  bearing  upon  the  subject 
matter  to  follow,  referring  the  reader  for  more  detailed  information  to  such  works 
as  Wilson's  "The  Cell  in  Development  aad  Inheritance;"  Mathews"  "Physiological 
Chemistry;"  Hammarsten's  "'Physiological  Chemistry;"  Gurwitsch's  "  Morpho- 
logic und  Biologic  der  Zelle;"  Hober's  " Physikalische  Chemie  der  Zelle  und  der 
Gevvebe;"  Hamburger's  "Osmotischer  Druck  und  lonenlehre;"  Loeb's  "Dynamics 
of  Living  Matter;"  Oppenheimer's  "Handbuch  der  Biochemie;"  and  Bottazzi, 
"Handbuch  der  vergl.  Physiologic,"  Vol.  I,  for  general  discussion,  and  to  the  most 
important  monographs  for  treatment  of  special  points. 
2  17 


■18     •  '-'    •  tk^'CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

■found  in  ■  plants,' iti  which  the  prominent  feature  is  the  hmiting  wall, 
forming  a  cell  to  enclose  a  fluid  content.  In  most  instances  the 
"cell"  answers  better  to  the  definition,  "a  mass  of  protoplasm;"  but 
usage  makes  language,  and  no  possible  confusion  can  arise  from  the 
prevailing  universal  use  of  the  original  term,  except,  perhaps,  that 
the  term  is  prone  to  carry  with  it  the  thought  of  the  walls  of  the 
cell  being  much  more  prominent  than  they  really  are.  This  is  not 
so  unfortunate  a  result,  perhaps,  for,  as  we  shall  see  later,  the  limiting 
surfaces  of  the  cell,  even  when  too  thin  to  be  readily  demonstrable, 
may  plaj^  a  much  more  important  part  in  cell  chemistry  than  their 
appearance  indicates. 

The  morphological  division  of  the  cell  into  cell  wall,  cytoplasm, 
nucleus,  and  nucleolus  can  hardly  be  followed  out  chemically,  for  if 
we  surmount  to  some  extent  the  difficulties  in  the  way  of  studying  the 
different  portions  separately,  we  find  that  the  differences  between  them 
are  rather  quantitative  than  qualitative.  And,  furthermore,  how- 
ever different  the  cells  of  one  organ  or  tissue  may  appear  from  those  of 
another  organ  or  tissue  under  the  microscope,  when  analj^zed  by  the 
chemical  methods  at  present  at  our  cUsposal  we  find  the  differences 
very  slight  indeed.  Certain  substances  are  found  in  every  living  cell, 
and  in  quantities  usually  not  greatly  dissimilar;  hence  they  are  as- 
sumed to  be  the  most  important  constituents  of  protoplasm,  and  are 
sometimes  called  the  primary  constituents  of  the  cell.  Manj^  other 
secondary  constituents  may  also  be  present,  some  of  which  are  so 
nearly  universal  that  we  are  not  sure  but  that  they  really  are  primary 
cell  components;  such  are  fat  and  glycogen.  Others  are  characteris- 
tics of  certain  cells,  such  as  melanin  and  keratin,  or  specific  products  of 
cell  metabolism,  such  as  mucin  and  the  specific  enzymes.  The  great 
histological  and  chemical  differences  existing  between  different  tis- 
sues depend  often  on  these  secondary  products,  as  in  fat  tissue  and 
squamous  epithelium;  or  upon  the  intercellular  substance,  as  with 
connective  tissue,  cartilage,  bone,  etc.,  which  may  be  looked  upon  as 
products  of  cell  activity. 

Protoplasm,  as  the  term  is  generally  used,  includes  the  various 
primary  constituents  with  the  fluids  permeating  or  dissolving  them, 
but  does  not  include  the  more  conspicuous  secondary  constituents, 
such  as  fat  droplets,  pigment  granules,  etc.,  nor  the  cell  membrane 
when  such  exists.  Evidently  it  is  a  vcrj''  indefinite  term,  to  be  avoided 
as  much  as  possible,  particularly  because  of  the  confu.sion  as  to  whether 
it  includes  the  nucleus  or  not,  different  authors  differing  in  this  respect 
in  their  usage  of  the  word. 

CHEMISTRY  OF  THE  ESSENTIAL  CELL  CONSTITUENTS 

To  enumerate  the  jirimary  or  essential  constituents  of  the  cell  ab- 
solutely is  not  possible,  for  the  rapid  advances  in  chemistry  may 
alter  all  classifications  without  warning,  but  i)i;u'tica11y  th(\v  may  be 


CHEMISTRY  OF  riiOTEINS  10 

grouped  iiiulcr  tho  h('a(liii|!;.s  of  i)rotc'iiis,  lipins,  carbohydrates,  salts, 
and  water,  and  no  attempt  will  i)o  made  to  give  here  more  than  the 
most  essential  features  concerning  each. 

Proteins  - 

In  the  last  few  years  we  have  obtained  something  approaching  a  scientific 
uuderstandinp;  of  the  chemical  nature  of  this  great  group  of  the  most  highly  com- 
])lcx  bodies  known  to  chemistry.  Our  information  has  been  obtained  almost  e.\- 
clusively  tiu-ough  studies  of  the  products  obtained  by  splitting  up  the  protein 
molecule,  for  as  yet  relatively  little  has  been  accomplished  through  synthesis. 
Proteins  can  be  decomposed  by  the  action  upon  them  of  acids  or  alkalies  in  various 
concentrations,  by  sui)erhcatcd  steam,  by  digestive  ferments,  and  by  bacteria. 
The  products  obtained  in  these  different  waj's  arc  not  all  the  same,  for  some  sub- 
stances may  be  formed  by  oxidation,  reduction,  decomposition,  combination,  or 
condensation  of  the  various  products  of  simple  cleavage,  and  it  is  necessary  to 
distinguish  between  the  primary  cleavage  products  (those  which  exist  as  radicals 
within  the  molecule)  and  the  secondary  products  (those  not  existing  preformed  in 
the  molecule  but  formed  by  transformation  of  the  primary  products).  This  can 
usually  be  done,  and  it  is  found  that  so  far  as  the  primary  products  are  concerned, 
it  makes  little  difference  which  method  of  cleavage  (or  hydrolysis,  since  in  the 
si)litting,  water  is  combined  with  the  organic  substances)  is  used. 

At  first  the  proteins  split  up  into  compounds  still  possessing  many  of  the  fea- 
tures of  the  typical  protein  molecule,  such  as  albumoses  and  peptones,  and  these 
bodies  are  then  further  resolved  into  simpler  substances,  which  are  not  aggregates 
of  several  smaller  molecules  as  are  the  proteins,  and  which  can  be  obtained  in  pure 
crystalline  form.  No  matter  which  method  is  used  we  find  the  process  going 
through  these  stages,  and,  as  before  mentioned,  the  primary  crj'stalline  products 
obtained  are  practically  the  same  quantitatively  as  well  as  qualitatively.  Some 
methods,  e.  g.,  bacterial  decomposition,  however,  lead  in  the  end  to  more  profound 
or  different  decomposition  of  the  cleavage  products  into  secondary  substances. 
The  similarity  of  the  results  obtained  in  these  different  ways  indicates  that  there 
are  definite  lines  of  cleavage  in  the  protein  molecule  along  which  separation  takes 
place,  independent  of  the  nature  of  the  agency  at  work,  and  that  the  substances 
obtained  represent  the  "building  stones"  of  the  entire  molecide. 

These  substances  all  have  in  common  one  important  point:  each  one  is  an  acid, 
which  has  a  NH2  group  substituted  for  a  hydrogen  atom  on  the  carbon  nearest  the  acid 
radical  (the  a-position).  It  makes  no  difference  what  the  rest  of  the  radicals  are, 
whether  they  are  simple  chains  (leucine),  or  members  of  the  cyclic  or  aromatic 
series  (tyrosine),  or  sulphur-containing  bodies  (cystine),  without  exception  this 
relation  of  a  NH2  group  to  an  acid  radical  is  constant,  as  in  this  formula: 

NH2 

/ 
R— CH— COOH. 

Through  this  arrangement  every  one  of  the  constituents  of  the  protein  mole- 
cule is  provided  with  a  group  with  a  strong  basic  character  arid  a  group  with  a 
strong  acid  character,  and  hence  it  is  possible  for  them  to  unite  with  one  another 
in  indefinite  numbers,  and,  because  of  the  great  variety  of  individuals,  in  practi- 
cally an  infinite  number  of  combinations.  It  is  believed  that  it  is  in  just  this  way 
that  the  protein  molecule  is  built  up.  By  artificially  uniting  various  cleavage 
products  Emil  Fischer  succeeded  in  producing  large  molecules  made  up  of  several 
amino-acid  radicals  (called  by  him  "polypeptids")^  which  show  some  of  the  char- 
acteristics of  the  peptones,  and  this  is  the  nearest  that  investigators  have  yet  come 
to  synthesizing  a  protein  molecule.  The  union  is  accomplished  by  the  splitting 
off  of  water,  corresponding  to  the  addition  of  water  that  occurs  when  the  protein 

-  For  the  complete  literature  of  this  subject  see  "The  Chemical  Constitution 
of  the  Proteins,"  PHmmer,  London,  1917;  "The  General  Character  of  the  Proteins," 
Schryver,  London,  1912;  "The  Vegetable  Proteins,"  Osborne,  London,  1910  (all 
in  the  series  of  "Monographs  on  Biochemistrv").  Also  "The  Physical  Chemistry 
of  the  Proteins,"  T.  B.  Robertson,  New  York,  1918. 

3  Reviewed  by  Fischer,  in  Ber.  deut.  Chem.  Gesell.,  1906  (39),  530. 


20  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

molecule  undergoes  cleavage.     It  ms^y  be  illustratetl  Ijy  showing  tlie  formation  of 
the  simplest  polypeptid,  (jlycy  I  glycine. 

N'H2  0  XH2  O 


i 


CH2  — C—  I  OH  +  H  I  HN  — CH2— COOH   =   CH:  — C  — HN  —  CH>— COOH  +  HjO. 

(glycine)  (glycine)  (glycylglycine) 

For  these  reasons  it  is  believed  that  tlie  protein  molec^ile  consists  of  great  numbers 
of  ami  no-acid  groups,  combined  with  one  another  through  their  basic  and  acid  radicals, 
and  that  the  various  proteins  are  different  from  one  another  because  they  contain 
different  numbers  or  varieties  or  orders  of  combination  of  amino-acids.  For 
example,  the  globin  of  hemoglobin  yields  no  glycine  on  hydrolysis,  while  gelatin 
yields  IG.o  per  cent.  On  the  other  hand,  gelatin  is  free  from  tyrosine.  Some  of 
the  prolamins  (proteins  obtained  chiefly  from  spermatozoa)  yield  as  high  as  58 
to  84  per  cent,  of  arginine,  while  the  simpler  amino-acids  with  but  one  N  (mono- 
amino-acids)  are  scanty,  and  most  varieties  are  lacking. 

It  will  be  noticed  that  when  two  amino-acids  unite,  as  seen  in  the  formation  of 
glycylglycine,  an  acid  radical  and  a  basic  radical  are  still  left  free.  In  this  may  be 
seen  the  e.xplanation  of  the  peculiar  amphoteric  nature  of  proteins.  As  long  a? 
these  two  groups  are  free  the  proteins  can  combine  with  either  acids  or  bases,  a? 
they  are  well  known  to  do,  and  hence  they  react  as  either  acids  or  bases  under  dif- 
ferent conditions. 

It  must  not  be  imagined  that  the  structiu-e  of  the  complete  molecule  is  simply 
a  long  straight  chain  of  amino-acids  joined  only  in  tlie  same  way  as  are  the  com- 
ponents of  glycylglycine.  The  existence  of  the  diamiuo-acids.  of  the  benzene 
rings,  of  hydroxyl  groups,  (as  in  serine  or  t\'rosine),  of  ring  compounds,  (as  pyrroli- 
dine carboxylic  acid),  of  substances  with  two  acid  groups,  (as  glutaminic  and  aspartic 
acid),  adds  complications  to  the  formation  until  it  is  impossible  to  estimate  just 
how  all  the  various  building  stones  may  be  arranged.  We  must  bear  in  mind  the 
size  of  the  protein  molecule,  which  Hofmeister  has  estimated  (for  serum  albumin) 
as  having  a  molecular  weight  of  10,1GG,  and  for  hemoglobin  the  molecular  weight 
has  been  estimated  at  16,669.  Within  such  a  "giant  molecule"  there  is  room  for 
variety  almost  beyond  comixitation. 

The  Proteins  of  the  Cell. — By  physiological  chemists  proteins 
are  clas.sified  into  simple  proteins,  of  which  egg  and  serum  albumin  are 
types;  and  compound  proteins,  which  are  characterized  by  having 
some  special  non-protein  group  which  can  be  split  off,  leaving  behind 
a  characteristic  protein  residue,  e.  g.,  nucleo-proteins,  glyco-proteins. 
As  primary  cell  constituents  the  following  varieties  of  proteins  may 
be  mentioned;  albumin,  globulin,  nucleo-protein,  nucleo-albiunin  or 
phospho-protein,  and  insoluble  pi'oteins.  At  one  time  it  was  thought 
that  cytoplasm  consisted  chiefly  of  albumin,  like  white  of  egg.  but 
we  now  know  that  this  forms  but  a  small  part  of  the  cell  proteins, 
often  occurring  only  as  traces.  It  is  held  by  some  that  true  albumin 
OcciH-s  only  as  a  building  or  intermediate  cleavage  product  of  the 
more  complicated  forms  of  cellular  proteins,  and  is  itself  of  relatively 
slight  importance  in  cell  life,  not  participating  in  cliemical  changes 
except  as  a  food-stuff. 

Albumins  arc  characterized  chiefly  by  tiieir  greater  solubility  in  water,  and  in 
being  less  easily  j)rccipitated  tluin  most  proteins.  Th(>y  seem  to  be  a  fundnmcntal 
type  of  proteins.  The  tlircc  forms  of  albumin  that  ha\('  ix'cii  dc^i-rilu'd  in  animal 
tissues  or  jjroducts  arc  egg-albumin,  lactalbumiu  of  milk,  aiul scrum  albumin;  pro- 
hal>ly  cell  albumin  is  most  closely  related  to  the  last,  and  what  has  been  described 
as  cell  albumin  is  perhaps  in  many  cases  but  .serum  albumin  that  has  been  imper- 
fect I  v  icmoved. 


CIII'MlSriy'Y  OF  I'h'oTKIXS     '  21 

Globulins  also  oocur  in  nil  cells,  hut  in  small  amounts  in  most  animal  ceils 
except  llie  muscles,  whose  cliief  proteins  helonp;  to  tliis  or  a  closely  related  Kroup. 
Tiie  globulins  are  quite  similar  to  the  albumins,  so  that  there  is  really  no  sliarp  line 
between  the  two  j!;roups.  Their  insolubility  in  water  separates  them  from  albu- 
mins, and  their  solubility  in  dilute  lunitral  salt  solutions  from  the  more  complex 
proteins.  An  important  feature  of  tli(>  f^loljulins  is  the  low  temperature  at  which 
they  coagulate — some  so  low  that  IIallil)urton'  believes  it  possible  that  they  may 
be  coagulated  within  the  cells  during  high  fevers. 

Hammarsten  has  long  maintained  that  simple  proteins  form  a  relatively  in- 
significant part  of  the  cytoplasm,  in  opjiosition  tf)  the  once-prevalent  view  that 
the  nucleo-proteins  were  limited  to  tlie  nucleus,  and  that  the  cytoplasm  was  chiefly 
albumin  and  globulin.  'Plie  general  trend  of  opinion  as  influenced  by  the  result.s 
of  researches  has  been  favorable  to  his  contentions,  and  we  shall  jjrobably  not  be 
far  wTong  in  accepting  his  statement  that — "The  chief  ma.ss  of  the  protein  sub- 
stances of  the  cells  does  not  consist  of  proteins  in  tlie  ordinary  sen.se,  but  consists 
of  more  complex  phospliorized  l^odies,  and  that  the  globulins  and  albumins  are 
to  be  considered  as  nutritive  materials  for  the  cells  or  as  destructive  products  in 
the  chemical  transformation  of  the  protoplasm."^ 

Nucleo-proteins  are  considered  to  be  the  most  important  constituents  of  the 
cell,  botli  in  quantity  and  in  relation  to  cell  activity.  In  structure  the  nucleo- 
proteins  are  very  complex,  as  indicated  by  the  difTerent  products  yielded  on  hydro- 
lytic  cleavage  of  the  molecule.  Furthermore,  there  are  many  varieties,  depending 
both  upon  the  nature  and  proportions  of  the  component  parts.  They  may  be 
described  as  consisting  of  two  primary  constituents — (1)  nucleic  acid  and  (2)  a 
protein  body,  in  chemical  combination  with  each  other  like  a  salt.  The  term 
itiicleic  acid  covers  a  large  group  of  substances,  which  are  characterized,  on  the 
one  hand,  by  their  frequent  occurrence  bound  with  proteins,  and,  on  the  other 
hand,  by  their  yielding  phosphoric  acid  and  purine  bases,  pyrimidines  and  pentoses 
or  hexoses  on  cleavage.  Diagranimatically  the  manner  of  cleavage  of  the  nucleo- 
proteins  may  be  indicated  as  follows:  * 

Nucleo-protein 

.  /\ 
nuclein^        protein 


nucleic  acid  protein 


phosphoric  acid         purine  bases,  pyrimidines  and  carbohydrates. 

In  the  cell  the  nucleo-proteins  probably  exist  partly  as  solid  structures,  c.  (j., 
the  chromatin  framework  of  the  nucleus,  and  partly  dissolved  in  the  plasma.  An 
interesting  phenomenon  is  the  alteration  in  the  chromatin  nucleo-proteins  during 
cell  division,  when  they  seem  to  lose  part  of  the  combined  protein  and  approach 
more. nearly  pure  nucleic  acid — just  as  inorganic  salts  occur  with  the  acids  and 
bases  saturating  each  other  more  or  less  incompletely,  e.  g.,  mono-,  di-,  and  tri- 
basic  phosphates.  In  this  we  have  a  chemical  explanation  of  the  intensity  of  the 
staining  of  dividing  nuclei  by  basic  dyes." 

Phosphoproteins  resemble  nucleo-proteins  to  the  extent  that  they  also  yield 
phosphoric  acid,  and  are  somewhat  similar  in  solubility  and  digestibility.  They 
are  essentially  different,  however,  in  that  they  do  not  yield  nucleic  acid  or  purine 
bases  on  cleavage.  Probably  members  of  this  group  are  also  constant  components 
of  cells. 

Glycoproteins  (or  gluco-prnl(in>i)  and  phof^pho-c/lycnproteins  are  also  believed  to 
occur  frequently  or  constantly  in  protoplasm.  They  are  compounds  of  proteins 
with  a  sugar  or  sugar-like  group,  which  probably  usually  contains  nitrogen,  thus 
differing  from  the  ordinary  hexoses  and  pentoses. 

'  Halliburton  and  jNIott,  Archives  of  Neurology.  1903  (2),  727;  also  .see  Halli- 
burton's "Chemistry  of  Muscle  and  Nerve." 

'See  Kossel,  Munch,  med.  Woch..  1911  (58),  65. 

*  Probably  nuclein  should  be  considered  as  merely  one  variety  of  nudeoprotcin, 
with  less  protein  than  the  other  varieties. 

'  The  chemistry  of  the  nucleo-proteins  is  discussed  in  the  chapter  on  Trie  Acid 
Metabolism  and  Gout,  Chap,  xxiii. 


22  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

Insoluble  proteins,  or  bodies  resembling  the  coagulated  proteins  in  their  lack  of 
solubility  in  various  fluids,  are  left  behind  after  the  other  proteins  have  been  ex- 
tracted from  the  cells.  Their  significance  is  not  known :  whether  to  a  large  extent 
artificially  produced  or  whether  a  normal  structural  element  of  the  cell. 

Fats  and  Lipoids  (Lipins)^ 

Lipoids  is  a  term  in  common  use  but  of  indefinite  significance; 
most  usually  it  comprehends  the  intracellular  substances  which  are 
soluble  in  ordinary  fat  solvents,  but  which  are  not  simple  fats  or  fatty 
acids,  lecithin  and  cholesterol  being  the  most  important  of  the  lipoids. 

For  the  entire  group  of  fats  and  lipoids  the  term  lipins  has  been  pro- 
posed by  Gies  and  Rosenbloom.^  Lipoids  and  ordinary  fats,  that  is, 
lipins,  occur  in  all  cells,  but  their  demonstration  is  not  always  readily 
possible.  The  microscopic  appearance  of  a  cell,  even  when  special 
stains  for  fat  are  used,  gives  no  correct  idea  of  the  amount  of  lipins 
actually  present.  Thus  normal  kidneys  contain  15  to  18  per  cent, 
in  their  dry  substance,  but  none  of  this  can  be  detected  readily  with 
the  microscope.  A  kidney  which  seems  microscopically  the  site  of 
marked  fatty  degeneration  may  show  no  more  fat  when  examined  chem- 
ically than  a  normal  kidney,  which  in  section  appears  to  be  quite  free 
from  fat.  This  is  because  some  of  the  intracellular  fat  is  so  bound, 
chemically  or  physically,  with  the  proteins  that  it  cannot  be  seen,  nor 
can  it  be  stained  by  the  dyes  ordinarily  used  for  that  purpose;  only 
when  degenerative  changes  of  certain  kinds  have  liberated  it  from  com- 
bination does  it  become  visible  and  stainable  bj^  ordinaiy  methods 
(Rosenfeld).  By  the  special  fixation  method  of  Ciaccio  the  fatty  com- 
pounds of  even  normal  cells  may  be  made  stainable  (Bell),^°  showing 
that  the  so-called  masked  fat  is  really  in  a  not  altogether  invisible  form. 
Whether  the  intracellular  fat  has  any  function  other  than  that  of 
serving  as  a  food-stuff  is  not  known,  but  there  can  be  no  question  of 
the  importance  of  the  phosphorized  fats,  or  phospholipins. 

Phospholipins  are  primary  cell-constituents  and  are  probably  important  both 
in  metabolism  and  physically.  Hammarsteu  regards  them  as  concerned  in  the 
ijuilding  up  of  the  nucleus.  As  will  be  shown  later,  manj-  of  the  most  essential 
physical  properties  of  the  living  cell  depend  upon  the  presence  in  it  of  lipoids,  of 
which  phosphatids  are  apparently  the  chief.  Of  the  ether-soluble  substances  in 
the  heart,  for  example,  09  to  70  per  cent,  are  phosphatids,  8  per  cent,  of  the  dry 
weight  of  the  myocardium.  Many  different  substances  have  been  described  as 
phosphatids,  but  the  chemical  identity  of  but  few  is  sufficiently  established.  Of 
these  the  most  imjjortant  are  lecithin  and  ccphalin,  which  are  most  intimately 
associated. 

There    are    several    possible  varieties  of  lecithin,  depending  upon  the  fatty 

*  Full  discussion  in  "Lecithin  and  Allied  Substances  (The  Lipins),"  by  Hugh 
MacLean,  Monographs  on  iJiociliemistry,  London,  1918. 

"  MacLean  uses  "li))in"  to  include  "subslances  of  a  fat-like  nature  yielding  on 
hydrolysis  fatty  acids  or  derivatives  of  fatty  acids  and  containing  in  their  nu)lecule 
either  N,  or  N  and  1'.  As  there  is  need  for  a  term  covering  the  fats,  phospliatids, 
cholesterols  and  related  Ixxlies,  the  suggestion  of  C.ie.s  .-uid  lUi.senbloom  is  followed 
for  tlie  i)resent  in  tliis  i)ook,  and  tin*  word  lipin  used  witli  the  broader  significance. 

">  Jour.  Med.  lies.,  1911  (1-9),  SiW. 


FATS  AND  LI  poms  23 

acid  radical  they  contain.  The  assumed  structural  formulaof  one  lecithin,  stearyl- 
olcyl  lecitliin,  is  as  follows: 

CH2 — O — C18 — HssO 

I 
CH— O— Ci,— H,30 

I 
CH2— 0— PO— OH 

I 
0— CHo— CH2— N  =  (CHa),. 

OH 

It  differs  from  ordinary  fats,  therefore,  in  having  two  special  groups,  one  the  phos- 
phoric acid,  the  other  the  choline  radical,  w^hich  last  may  be  of  some  importance 
in  pathological  processes.  In  its  phj^sical  properties  it  is  quite  similar  to  the 
ordinary  fats,  although  it  forms  even  finer  emiilsions  in  water,  which  are  practically 
colloidal  solutions  (W.  Koch). 

Cephalin  diiTers  in  having  for  the  base  amino-ethyl  alcohol  (XH0CH2CH2OH) 
instead  of  choline,  and  is  probably  as  widely  spread  in  the  tissues  as  lecithin. 1^ 
It  has  been  held  by  some  that  there  are  many  phospholipins,  which  may  be  speci- 
fic for  different  cells,  tissues  and  species,  but  it  seems  more  probable  that  these 
supposed  specific  lipoidal  substances  are  merely  mixtures  of  lecithin,  cephalin  and 
their  derivatives  in  varying  proportions  (Levene).^^ 

Cholesterol,  which  is  another  lipoid,  is  nearly  as  universally  present  as  leci- 
thin, it  exists  both  free  and  in  combination  ^\•ith  fatt}'  acids,  for  cholesterol  is 
an  alcohol  and  not  at  all  similar  to  the  fats  chemically,  although  very  similar 
physically.  The  empirical  formula  is  C27H4SOH  or  C27H46OH,  and  it  is  related 
to  the  terpenes.  It  seems  to  be  relatively  inert  chemically,  and  therefore  is 
probably  important  only  because  of  its  effect  on  the  phj'sical  properties  of  the  ceUs. 
By  some  it  is  considered  to  be  a  decomposition  or  cleavage  product  of  the  proteins, 
which  is  in  accordance  'ndth  its  abundance  in  masses  of  old  necrotic  tissue,  e.  g., 
atheromatous  masses,  old  infarcts,  and  old  exudates. 

Doubly  Refractive  Lipoids  and  Myelins. '^ — In  practically  aU  normal  tissues 
there  are  present  droplets  of  lipoid  nature  which  are  characterized  bj'  sho-ndng 
prominent  crosses  when  examined  with  crossed  Nicol  prisms  (anisotropic),  the 
adrenal  and  corpus  luteum  containing  them  most  abundantly.  Chemically  they 
seem  to  be  mixtures  of  various  lipoids  in  inconstant  proportions,  but  probably  the 
anisotropic  character  is  most  usuallj'  dependent  upon  the  presence  of  cholesterol 
esters.  The  term  myelin  was  first  appUed  by  Virchow  to  pecuHar  fatty  substances 
found  in  various  normal  and  pathological  tissues,  because  they  showed  physical 
characters  similar  to  those  of  the  myeUn  substance  of  nerves,  but  as  many  of  these 
substances  are  doubly  refractive,  or  can  be  easily  made  so,  some  authors  use  the 
term  myelin  as  if  it  were  synonymous  with  doubly  refractive  Hpoids.  There  are, 
however,  myelins  which  are  not  always  doubly  refractive,  and  also  doubly 
refractive  hpoids  w^hich  do  not  swell  up  in  water  to  form  myeUn  figures,  etc.,  as  is 
characteristic  of  true  myeUns.  Chemically,  however,  the  mj^ehns  and  doubly 
refractive  substances  are  probably  related,  consisting  of  mixtures  of  cholesterol, 
cholesterol  esters,  phospholipins  and  perhaps  soaps,  in  varying  proportion.  They 
will  be  considered  further  in  discussing  Fatty  Metamorphosis,  Chap.  XVI. 

Carbohydrates 

The  third  great  class  of  food-stuffs,  the  carbohydrates,  is  represented  in  the  ceU  by 
pentoses  and  hexoses  combined  with  proteins  and  with  lipoids,  and  also  by  glycogen, 
which  exists  free.  Glycogen  is  a  difficult  substance  to  isolate  in  minute  quantities 
and,  therefore,  although  it  is  not  found  in  all  cells  by  our  present  methods,  yet  it 
may  well  be  that  it  is  a  constant  constituent  of  the  protoplasm.     There  is  no  e\a- 

"  Koch  and  Woods,  Jour.  Biol.  Chem.,  1905  (1),  203. 

12  Jour.  Biol.  Chem.,  1919  (39),  S3. 

"See  Adami,  Join:.  Amer.  Med.  Assoc,  1907  (48),  463;  Karwdcka,  Ziegler's 
Beitr.,  1911  (50),  437;  Schultze,  Ergebnisse  Pathol.,-1909  (13,  pt.  2),»253;  Bang, 
Ergebnisse  Physiol.,  1907  (6),  131;  1909  (8),  463. 


21  THE  CHEMISTRY  AMJ  PHYSICS  OF  THE  CELL 

dence,  however,  that  it  is  anything  more  than  a  source  of  heat  and  energy  to  the 
cell.  Its  properties  and  occurrence  will  be  considered  more  fully  in  the  discus- 
sion of  glycogenic  infiltration.  Since  glycogen  is  formed  from  dextrose  and  is 
constantly  breaking  down  into  dextrose,  it  is  probable  that  the  latter  is  also  con- 
stantly present  in  the  cells. 

Inorganic  Substances 

Up  to  this  point  the  substanf es  of  the  cytoplasm  that  have  been  discussed  have 
all  been  organic  compounds  which  do  not  naturally  exist  independent  from  living 
or  once  living  cells,  yet  the  inorganic  substances  of  the  protoplasm  are  also  of  vital 
importance.  As  Mann  says,  "so-called  pure  ash-free  proteins  are  chemically 
inert,  and,  in  the  true  sense  of  the  word,  dead  bodies.  What  puts  life  into  them 
is  the  presence  of  electrolytes."  The  various  salts  of  potassium,  sodium,  calcium, 
magnesium,  and  iron  which  all  cells  contain  do  not  exist  merely  dissolved  in  the 
water  of  the  cell,  but  in  part  they  are  comlnned  with  the  organic  ccmstituents 
of  the  protoplasm.  They  are  not  combined  as  simple  additions  of  the  salts  to 
the  proteins;  but  io)is,  both  anions  and  cations,  are  united  in  chemical  combina- 
tion to  the  large  protein  molecule  (ion-proteins)."  Possibly  the  proteins  partici- 
pate in  vital  chemical  processes  only  as  ion  compounds  with  inorganic  elements. 
It  is  extremely  difficult,  indeed  almost  impossible,  to  secure  proteins  entirely  free 
from  inorganic  substances  (ash-free  proteins).  The  fact  that  inorganic  substances 
are  held  in  the  cells  cliemically  rather  than  by  simple  diffusion  into  them  from  the 
surrounding  fluids  is  shown  by  the  great  difference  in  the  proportions  of  various 
salts  in  the  cells  and  in  the  extra-cellular  fluids.  Thus  potassium  is  nearly  always 
much  more  abundant  in  the  cells  than  in  the  tissue  fluids,  while  sodium  is  more 
abundant  in  the  fluids.  Piiosphoric  acid  is  also  more  abundant  in  the  cells,  and 
chlorin  in  the  plasma.  In  cells  iron  seems  to  exist  chiefly  in  combination  with  the 
nucleo-protcins.  '^ 

THE  PHYSICAL  CHEMISTRY  OF  THE  CELL  AND  ITS  CONSTITUENTS'" 

From  the  standpoint  of  physical  chemist ly  the  cell  consists  of  a 
collection  of  colloids  and  crystalloids,  electrolytes  and  non-electrolytes, 
dissolved  in  water,  in  lipoids,  and  in  each  other,  surrounded  by  a  semi- 
permeable membrane,  and  perhaps  subdivided  by  similar  membranes 
or  surfaces.  Physical  chemical  jjrocesses,  as  we  shall  see  later, 
play  an  all-important  part  in  the  life  phenomena  of  the  cell,  and  there- 
fore some  space  may  be  occupied  profitabl}^  in  explaining  the  nature 
of    these  changes  and  of  the  .substances   that    participate  in   them. 

Crystalloids  and  their  Properties 

Crystalloids,  or  substances  that  tend  under  favorable  conditions 
to  form  crystals,  and  which  diffuse  readily  through  most  diffusion 
membranes,  form  a  relatively  small  part  of  the  total  mass  of  the  cell, 
but  they  are  fully  as  essential  as  the  colloids.  The  chief  representa- 
tives of  this  groui)  that  are  found  usually  or  constantly  in  the  cell  are 
the  inorganic  salts,  sugar,  and  the  innumerable  decomposition  products 
of  the  proteins,  including  particularly  urea,  creatine,  purine  bases, 
amino-acids,  etc.  Most  of  these  are  by  no  means  so  characteristic 
of  living  things  as  are  the  colloids,  sometimes  occurring  (juite  inde- 

"SeeJ.  Loci),  Science,  191't  (SO),  4:{<). 

"  See  Macallum  on  Microchemistry,  lMgei)nisse  Piiysiol.,  1908  (7),  552. 
"See  Hayliss,  "Principles  of  (Jciieral  Physiology,"  London,  1915,  for  a  more 
extensive  discussion  of  these  topics. 


CRYSTALWIDS  25 

peiHlciif  ly  ot"  a  (•cUiilai'  origin,  which  the  proteins  never  do.  Tlie  inoi- 
ganic  salts  in  i)arti('iihir  seoni  (luilc  i'oi-eifj;n  to  H\inji  processes,  and 
as  they  enter  and  leave  the  body  i)ractieally  unchanged  they  are 
evidently  not  a  source  of  energy  through  chemical  change.  Their 
importance  to  the  cell  lies  almost  entirely  in  their  physical  or  physico- 
cliemical  properties.  The  organic  crystalloids,  although  of  nutri- 
tional value,  also  have  ])hysical  jiropei-ties  in  some  respects  similar  to 
those  of  the  inorganic  crystalloids,  and  therefore  to  this  extent  they 
exert  similar  influences,  but  the  essential  difference  between  the  organic 
and  the  inorganic  crystalloids  is  that  all  the  latter  are  electrolytes, 
while  many  of  the  organic  crystalloids  that  occur  in  cells  are  non- 
electrolytes.  The  importance  of  this  distinction  lies  not  in  the  utility 
or  non-utility  of  these  substances  as  conductors  of  electrical  currents 
in  the  ordinary  sense,  but  rather  on  the  existence  of  those  properties 
which  determine  their  conductive  al)ility.  Electrical  conductivity  is 
an  index  of  ionization,  and  upon  ionization  depends  the  chief  influence 
of  the  electrolytes  upon  vital  activities.  The  importance  of  this 
process  of  dissociation  or  ionization  lies  in  the  fact  that  with  most 
substances  no  chemical  reaction  can  occur  while  the  substance  is  in  the 
non-ionized  state.  The  chemical  properties  of  ionizable  substances 
are  produced  largely  by  the  ions  they  liberate  on  dissociation.  As  a 
consequence,  the  physiological  effects  of  electrolytes  are  due  to  their 
ionic  condition,  and  through  the  ions  that  are  present  in  the  cell  many 
of  its  various  chemical  processes  are  brought  about.  Not  all  substances 
ionize  with  the  same  readiness,  which  causes  a  great  difference  in  their 
properties.  The  reason  that  acetic  acid  is  a  weaker  acid  than  hydro- 
chloric acid  is  that  it  does  not  ionize  to  such  an  extent,  and  so  a  cor- 
responding ciuantity  does  not  introduce  as  large  a  number  of  hydrogen 
ions  into  a  solution.  Larger  molecules,  as  a  rule,  ionize  less  than  smal- 
ler ones  of  similar  nature,  e.  g.,  stearic  acid  ionizes  less  than  acetic  acid 
and  therefore  is  a  weaker  acid.  Likewise  the  properties  of  a  substance 
which  depend  upon  its  ions  will  be  less  marked  when  it  is  in  a  solvent 
that  produces  little  ionization.  For  example,  bichloride  of  mercury 
owes  its  antiseptic  properties  to  the  Hg  ions  that  it  sets  free  when  in 
solution.  It  is  well  known  that  solutions  of  mercury,  and  for  that 
matter  most  other  antiseptics,  are  much  less  actively  germicidal  in 
alcohol  than  when  in  water,  because  their  ionization  is  less  in  alcohol; 
and  the  germicidal  properties  decrease  as  the  proportion  of  alcohol 
increases,  until  the  germicidal  effect  of  the  mixture  is  no  greater  than 
that  of  alcohol  alone  in  the  same  strength. 

If  we  had  no  electrolytes  in  the  cell,  electric  charges  could  not  be 
carried  about  in  it,  and  hence  chemical  reactions  could  not  occur. 
It  is  this  fact  that  makes  the  inorganic  salts  of  such  vital  importance 
to  the  cell  life.  To  repeat  Mann's  words,  it  is  the  electrolytes  that 
put  life  into  the  proteins.  Water  itself  is  almost  absolutely  non-disso- 
ciated, and  proteins  so  little  that  for  some  time  it  was  doubted  if  they 


26  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

really  did  ionize.  Probably  all  soluble  substances  do  dissociate  to  a 
certain  minimal  degree,  but  it  is  so  slight  for  most  of  the  constituents 
of  the  cell  except  the  inorganic  salts  (the  organic  acids  and  alkalies, 
and  a  few  dissociable  organic  products  of  protein  metabolism,  occur  in 
such  insignificant  amounts  as  to  be  almost  negligible)  that  without 
them  there  would  be  little  chemical  activit}^  possible,  and  hence  life 
would  be  absent  or  at  a  very  low  ebb  indeed.  As  before  mentioned, 
the  inorganic  salts  probably  exist  in  the  cell  not  only  as  salts,  but  also, 
and  perhaps  chiefly,  as  ions  and  ionic  compounds  with  the  cell  proteins. 

Many  applications  of  the  facts  and  theories  of  ionization  have  been 
made  in  physiology  and  a  few  applications  have  also  been  made  in 
pathology,  especially  the  relation  of  ions  to  edema,  to  diuresis  and 
glycosuria,  and  also  to  problems  of  immunity.  No  attempt  will  be 
made  here  to  go  further  into  the  observations  and  theories  concerning 
ionization  or  its  role  in  physiology,  but  for  more  extensive  information 
as  well  as  for  the  complete  bibliographj^  the  works  mentioned  below 
may  be  referred  to.^'  The  applications  in  pathology  will  be  brought 
out  as  the  subject  under  discussion  in  subsequent  chapters  necessitates 
and  it  is  largely  to  facilitate  the  understanding  of  such  reference  that 
this  brief  summary  of  the  subject  of  ionization  has  been  introduced. 
In  the  same  spirit  we  take  up  the  subjects  of  diffusion  and  osmosis. 

Diffusion  and  Osmosis. — Although  the  non-electrolytes  do  not 
ionize  to  any  considerable  extent,  and  therefore  are  relatively  inactive 
chemically,  the  crystalloidal  non-electrolytes,  of  which  sugar  and  urea 
are  the  two  chief  examples  among  the  cell  constituents,  possess  in 
common  with  the  electrolytes  the  important  property  of  diffusion. 
By  this  process  the  exchange  of  chemical  substances  between  the  blood 
and  the  cell  is  brought  about,  by  it  the  chemical  composition  of  the 
different  parts  of  the  cell  and  between  different  cells  is  equalized, 
and  without  it  chemical  change  would  be  practically  impossible.  Dif- 
fusion occurs  most  simply  between  two  solutions  of  unlike  nature, 
or  between  a  solution  of  a  substance  and  the  solvent  alone,  when 
placed  directly  in  contact  with  one  another.  If  we  place  in  the  bot- 
tom of  a  cylindrical  vessel  a  solution  of  copper  sulphate  and  above 
it  some  water,  carefully  avoiding  mixing,  it  will  be  found  after  some 
time  that  the  fluid  has  become  equally  blue  throughout.  This  is 
brought  about  by  the  movement  of  the  dissolved  particles  which 
gradually  carries  them  through  the  entire  mass  of  iluid,  and  as  their 
migration  is  against  the  force  of  gravity,  they  evidently  accomplish 

"  "Physical  Chemistry  in  the  Service  of  Medicine,"  Wolfgang  Pauli,  transla- 
tion by  M.  H.  Fischer,  New  York,  1907.  "  Physikalische  Chemie  dor  Zelle  und 
der  Gewehe,"  Hober,  Leipzig,  1915.  "Osmotisohe  Druck  und  lonenlehre  in  den 
medicinischen  Wissenscliaften,"  Hamburger,  Wiesbaden.  "Studies  in  General 
Physiology,"  Loci),  University  of  Chicago  Press,  1905.  "Dynamics  of  Living 
Matter,"  Locb,  Columbia  University  Press,  New  York,  1906.  Spiro  and  J.  Loeb 
Oppenheimer's  "Handbuch  der  Biochemie,"  1908  (2),  1-141.  "Physical  Chem- 
istry of  Vital  Piienomena,"  McCIendon,  Princeton  Univ.  Press,  1917. 


DIFFCSION  AND  OSMOSIS  27 

work.  'Hiis  process  is  not  dependent  upon  ionization,  for  a  s(jluti(ni 
of  cane-sugar  or  of  urea  will  show  the  same  diffusion.  A  sohition  of 
l)rotein  or  other  colloid  does  so  nuich  more  slowly,  however,  indeed, 
([uite  imperceptibly. 

If  we  were  to  introduce  a  piece  of  filter-paper  between  the  water 
and  the  copper  sulphate  solution,  the  diffusion  would  go  on  the  same, 
the  pores  of  the  paper  permitting  the  passage  of  the  molecules  with- 
out hindrance.  If,  instead  of  filter-paper,  there  were  introduced  a 
sheet  of  some  substance  free  from  pores,  the  diffusion  would  be  much 
more  affected.  If  the  septum  was  of  such  a  nature  that  the  sub- 
stances in  solution  were  insoluble  in  it  (e.  g.,  glass),  diffusion  would 
of  necessity  stop;  but  if  it  were  something  in  which  the  solvent  or  the 
solute  was  soluble,  such  as  a  gelatin  plate,  then  these  substances  would 
dissolve  in  it,  and  diffusing  through  its  substance  escape  into  the 
fluid  on  the  other  side.  The  last  example  indicates  the  conditions 
afforded  in  the  animal  cell,  and  also  in  the  usual  laboratory  diffusion 
experiments  when  the  membrane  is  generally  either  an  animal  mem- 
brane or  a  parchment  paper,  both  of  which  are  composed  of  colloids. 
Crj^stalloids  are  generally  soluble  in  colloids  and  hence  pass  through 
such  diffusion  membranes;  colloids  dissolve  but  slightlj'  in  colloids, 
and  hence  they  do  not  pass  through  a  diffusion  membrane  readily, 
and  are,  therefore,  but  very  slightly  diffusible. 

The  process  of  diffusion,  if  uninterrupted,  always  continues  until 
the  solution  is  of  exactly  the  same  composition  throughout.  If  on  one 
side  of  the  diffusion  membrane  there  is  a  substance  that  passes  through 
the  membrane  rapidly,  and  on  the  other  a  substance  that  passes 
through  slowlj'  or  not  at  all,  there  will  soon  be  an  unequal  condition  on 
the  two  sides  of  the  membrane,  for  the  diffusible  substance  would  ac- 
cumulate in  equal  amounts  on  each  side,  while  the  non-diffusible 
would  remain  where  it  was.  On  one  side  there  would  then  be  more 
material  exerting  osmotic  pressure  than  on  the  other,  and  if  the  mem- 
brane were  flexible,  it  would  bulge  toward  the  opposite  side.  The 
pressure  is  supposed  to  be  due  to  the  bombardment  of  the  containing 
walls  by  molecules  or  ions  of  the  substances  in  solution,  and  hence  the 
more  molecules  and  ions  in  solution,  the  more  pressure.  When  equal 
numbers  of  particles  are  on  each  side  of  the  partition,  the  pressure  is 
equalized.  It  is  quite  possible  to  have  membranes  readily  permeable 
to  one  substance  and  almost  entirelj^  impermeable  to  another;  such 
membranes  are  called  semipermeable.  To  produce  osmotic  pressure 
it  is  not  necessary  that  the  membrane  be  absolutely  impermeable  to 
any  of  the  substances — it  may  only  be  relatively  less  permeable  for 
the  solute  than  for  the  solvent.  If,  for  example,  we  fill  a  parchment 
bag  with  concentrated  sugar  solution,  tie  up  the  top  tightly  and  throw 
it  into  water,  it  will  swell  up  rapidly  and  eventually  burst.  But  if  the 
parchment  is  in  the  form  of  a  tube,  open  at  the  top,  and  the  lower 
end  is  placed  in  water,  the  amount  of  fluid  inside  the  tube  will  in- 


28  THE  CHEMISTRY  AND  PHYSIOS  OF  THE  CELL 

crease  at  first,  but  eventually  the  sugar  will  diifuse  out  to  such  an 
extent  that  the  solution  is  of  the  same  concentration  inside  and  out- 
side of  the  tube,  and  the  column  of  fluid  will  again  become  of  equal 
height  on  both  sides.  These  results  indicate  that  the  water  passes 
through  the  membrane  more  rapidly  than  does  the  sugar,  but  that 
eventuall}^  tlu;  sugar  can  all  pass  through. 

Exactly  similar  conditions  exist  in  cells,  particularly  plant  cells. 
The  typical  cell  of  plant  tissue  consists  of  a  distinct  wall,  usually 
cellulose,  lined  internally  by  a  layer  of  protoplasm  which  incloses 
a  mass  of  aqueous  solution,  the  cell  sap.  containing  sugar  and  various 
other  solutes.  The  outer  wall  is  readil}-  permeable  b}'  water  and  by 
most  solutes,  whereas  the  protoplasmic  layer  inside  it  behaves  like  a 
semipermeable  membrane,  which  permits  water  to  pass  through 
readily  but  hinders  greatly  the  passage  of  most  solutes;  that  it  is 
somewhat  permeable  is  attested  by  the  fact  that  the  cell  sap  contains 
solutes  derived  from  the  external  fluids.  As  a  result  of  this  arrange- 
ment there  is  a  constant  tendency  for  the  cavity  of  the  cell  to  be 
distended  by  water  and  for  the  solutes  within  it  to  exert  their  con- 
siderable pressure  upon  the  cell  wall.  Because  of  the  strength  of  the 
cellulose  layer  the  cell  can  withstand  great  pressures  that  Avould 
tear  apart  the  tender  protoplasmic  layer  that  really  determines  the 
osmotic  pressure  that  causes  the  rigidity  or  turgor  of  plant  (-('lis, 
and  explains  the  ability  of  a  tender  green  shoot  to  hold  itself  up- 
right or  horizontal  in  the  air;  and  it  is  the  force  that  enables  growing 
roots  to  lift  great  stones  or  tear  apart  rocks  in  whose  clefts  they  grow. 
If  certain  plant  cells  are  placed  in  distilled  water,  the  pressure  may 
rise  to  such  an  extent  that  the  cells  burst,  and  it  was  through  studying 
this  phenomenon  that  Pfeffer  worked  out  the  basis  of  our  pn^sent 
knowledge  of  osmotic  pressure.  If  the  cell  is  placed  in  a  solution 
of  greater  concentration  than  its  cell  sap,  the  pressure  outside  will 
be  greater  than  that  inside  and  the  protoplasmic  meml>rane  will  be 
forced  away  from  the  cellulose  wall,  while  its  central  cavity  shrinks 
and  i)erhaps  disappears  entirely,  the  protoplasm  forming  a  ball  in  the 
center.  This  is  practically  what  occurs  when  a  plant  stem  is  cut 
and  it  "wilts" — the  water  is  removed  by  evaporation,  the  osmotic 
pressure  outside  the  cells  becomes  greater  than  that  inside,  and  the 
water  passes  out.  Likewise  when  a  plant  c(>ll  dies  the  turgor  is  lost 
because  the  membrane  becomes  ))ermeable,  and  so  pi'(>ssure  soon  be- 
conuis  the  sanu^  on  both  sides  of  the  cell  wall. 

In  animal  cells  the  wall  is  not  so  highly  developed  as  in  plants, 
nor  is  it  backed  up  b}^  a  rigid  material  like  celluIos(>;  indeed  for 
many  animal  cells  there  is  no  well-defined  wall  and  the  piotojilasm 
appears  to  be  naked.  Nevertlu^le.ss  the  behavior  of  tlu>  animal  cells 
indicates  that  they  do  possess  what  resiMubles  a  ('(>11  wall,  in  that 
they  behave  when  in  solutions  as  if  they  wei-e  surrounded  by  a  dif- 
fusion   membi'aTie.      The   degi-ee   to   which    piieiioineii;!    ot'   this   iiatui'e 


DIFFISIOX  AM)  OSMOSIS  29 

aic  sluiwn  vniic's  witli  (liflciciit  cells;  with  red  coiijusclcs.  I'oi-  example, 
the  osmotic  pressure  influences  arc  very  marked,  as  shown  by  the 
wrinkling  or  crenation  of  the  corpuscles  when  they  are  placed  in 
fluitls  of  higher  concentration  than  the  blood  plasma,  and  by  their 
swelling  and  disintegration  with  escape  of  the  hemoglobin  {hemoly- 
sin) when  they  are  put  into  distilled  water  or  solutions  of  less  con- 
centration than  the  plasma.  Other  tissue  cells  seem  to  undergo  more 
or  less  alteration  from  changes  in  the  osmotic  pressure  in  the  fluids 
surrounding  tiieni.  The  dift"usion  membrane  that  surrounds  the  cell 
is  generally  nut  well  defincul,  and  for  most  cells  seems  to  be  but  a 
surface  condensation  of  the  protoplasm,  perhaps  formed  through  the 
effects  of  surface  tension.  It  seems  probable  that  this  surface  dif- 
fusion membrane  contains  a  large  proportion  of  cell  lipoids,  i.e., 
cholesterol  and  phospholipins  (for  the  red  corpuscles  this  is  practically 
certain);  hence  substances  soluble  in  lipoids  penetrate  the  cell  read- 
ily, while  to  many  substances  insoluble  in  lipoids  the  cell  is  nearly 
or  quite  impermeable  (Overton).  Probably  the  wall  of  the  animal 
cell  is  not  so  nearly  semipermeable  as  is  that  of  the  plant  cell,  for 
nowhere  in  the  animal  bod}-  do  we  get  such  turgor  in  the  cells  as 
we  see  in  plant  tissues.  Lacking  a  cellulose  wall,  animal  cells  could 
not  develop  such  an  internal  pressure  without  rupturing  and  such 
a  process  of  rupturing  {plasmorrhexis,  plasmoptysis)  does  not  seem 
to  be  a  normal  occurrence  in  animal  tissues.  We  shall  be  most 
nearly  correct,  probably,  if  we  look  upon  the  animal  cell  as  possess- 
ing a  delicate  diffusion  membrane  at  its  surface,  through  which  water 
passes  more  readil}^  than  do  most  crj'stalloids,  and  through  which 
colloids  pass  almost  not  at  all,  but  the  exclusion  of  each  of  these  types 
of  substances  is  merel}^  relative  and  not  absolute.  AVithin  the  cell, 
also,  the  colloids  probably  exist  as  a  more  or  less  well-developed 
emulsion,  so  that  we  have  here  a  practically  limitless  amount  of 
surface  formation  all  through  the  protoplasm;  such  a  structure  could 
permit  the  endless  number  of  reactions  of  a  living  cell  to  go  on  side 
by  side  in  the  same  cell.  Stuches  b}'  G.  L.  Kite^^  seem  to  show  that 
all  of  the  protoplasm  has  much  the  same  relation  to  solutions  as  does 
the  external  layer  or  cell  membrane,  for  he  found  that  if  drops  of 
solutions  which  can  penetrate  a  cell  from  outside  be  injected  directly 
into  a  cell  they  diffuse  through  it,  but  substances  which  cannot  pene- 
trate from  outside  are  also  unable  to  diffuse  through  the  cell  after  they 
have  been  injected  into  it. 

Since  osmotic  pressure,  exactly  like  gas  pressure,  is  presumably 
produced  by  the  bombarding  of  the  w^alls  of  the  container  by  parti- 
cles in  the  solution,  the  amount  of  pressure  will  vary  in  proportion 
to  the  number  of  particles  present.  With  non-electroh^es,  such 
as  sugar  and  urea,  the  moving  particles  seem  to  be  mole- 
cules, and  so  a  solution  of  sugar  or  urea  will  produce  an  osmotic 

's  Amer.  Jour.  Physiol.,  1915  (37),  282. 


30  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

pressure  directly  proportional  to  the  number  of  molecules  it  con- 
tains. In  the  case  of  the  electrolytes,  however,  the  ions  produce 
pressure  as  well  as  the  molecules,  and  hence  an  electrolyte  in  solution 
will  produce  a  relatively  high  osmotic  pressure  as  compared  with  an 
equivalent  solution  of  a  non-electrolyte,  since  each  molecule  may  jaeld 
two  or  more  ions.  Colloids,  however,  exert  so  slight  an  osmotic 
pressure  that  it  is  difficult  of  detection;  this  probably  depends  on 
the  great  size  and  slight  motility  of  their  molecules.  In  the  many 
and  important  osmotic  processes  of  the  animal  organism,  therefore, 
the  colloids  take  no  part  except  in  helping  to  form  the  diffusion  mem- 
brane, and  in  preventing  the  diffusion  of  one  another. ^^  It  is  interest- 
ing to  consider  also  that  colloids  under  ordinary  conditions  do  not 
greatly  modify  the  diffusion  of  crystalloids  through  a  solution  con- 
taining both  classes  of  matter.  The  fact  that  a  cell  is  full  of  dis- 
solved colloids  does  not  seriously  affect  the  osmotic  properties  of  the 
intracellular  crystalloids,  provided  the  colloids  are  not  condensed 
in  such  a  way  as  to  form  diffusion  membranes.  But  as  all  the  cleav- 
age products  of  proteins  after  they  have  passed  the  peptone  stage  are 
crystalloids,  by  decomposition  of  the  intracellular  proteins  the  os- 
motic pressure  may  be  greatly  raised.  As  long  as  the  cell  is  living 
there  can  be  no  constancy  in  composition,  for  metabolic  processes, 
by  producing  from  proteins  that  have  no  osmotic  pressure  crystal- 
loidal  substances  that  do  have  osmotic  pressure,  cause  intracellular 
osmotic  conditions  to  be  continually  varying.  As  a  result,  streams 
of  diffusing  particles  are  moving  about  in  every  direction,  setting  up 
new  chemical  reactions  and  consequent  new  osmotic  currents.  The 
greater  the  difference  in  osmotic  pressure  between  a  cell  and  its 
environment,  and  between  the  different  parts  of  the  same  cell,  the  more 
powerful  the  osmotic  effects,  and  as  a  result  the  greater  the  capacity 
for  accomplishing  work. 

Indeed,  we  may  look  upon  cell  life  as  a  constant  attempt  at  the 
establishment  of  equilibrium,  both  chemical  and  osmotic,  ichich  is  nether 
achieved  because  the  move  towards  one  sort  of  equilibrium  is  always 
against  the  other.  All  the  food-stuffs — -fats,  carbohjairates  and  pro- 
teins— are  characterized  by  being  colloids  when  intact  and  crystalloids 
when  disintegrated,  thus: 

colloidal  proteins  «=^  crystjilloidal  aiiiino  acids 
colloidal  f!;lycoG;en  <=i  crystalloidal  siijiar 
nondilTiisil)Ie  fats  ^  dilTusihlo  soaps  and  {glycerol. 

In  consequence  of  this,  if  the  crystalloids  difTuse  from  the  blood  into  a 
cell  there  is  at  once  an  excess  of  this  end  of  the  equation,  and,  hastened 
by  the  intraccillular  enzymes,  partial  syntliesis  to  the   colloid  soon 

'"  Under  experimental  conditions  it  is  found  that  the  nature  of  the  nienibrane 
greatly  modifies  the  osmotic  jiressure;  for  if  a  ^iven  colloid  is  soluV^le  in  a  cer- 
tain mend)rane  and  a  certain  crystalloid  is  not,  the  colloid  will  diffuse  through 
the  membrane  wliile  the  crystalloid  is  held  back.  (Kaldenberj!;,  ,Iour.  Physical 
Chem.,  1900  (10),  111.) 


COLLOIDS  31 

occurs  to  establish  chemical  ('quili])riuin.  Chemical  changes  in  the 
crystalloids,  by  oxidation,  nuhiclion  or  hych'olysis,  upset  this  chem- 
ical ecjuihbrium,  and  hence  further  diffusion,  synthesis  and  hydrolysis 
continue,  one  upsetting  the  other  continuously.  If  equilibrium  were 
established  we  should  have  no  further  reactions,  and  the  cells  would 
be  inactive.  The  constant  upsetting  of  the  equUihrium  is  what  con- 
stitutes cell  life. 

The  relation  of  osmotic  pressure  and  osmosis  to  physiological  prob- 
lems is  only  beginning  to  be  studied.  It  is  apparent  that  they  must 
be  of  essential  importance  in  absorption  from  the  ahmcntary  canal, 
in  absorption  and  oxciction  betAveen  the  cells  and  the  blood  stream, 
and  in  secretion  by  glandular  organs;  but  it  is  also  certain  that  they 
are  no  less  important  in  all  the  less  obvious  chemical  and  physical 
processes  of  the  cell. 2°  In  pathological  processes  osmotic  pressure 
may  play  an  equally  important  role,  and  the  facts  discussed  in  the 
prececUng  paragraphs  will  be  alluded  to  frequently  in  subsequent 
chapters. 

COLLOIDS-i 

Since  Graham  in  1861  studied  the  differences  between  the  sub- 
stances that  chd  or  did  not  diffuse  reachly  through  animal  or  parch- 
ment membranes,  soluble  substances  have  been  classified  in  the  two 
main  groups  of  colloids  and  crystalloids,  which  distinction  Graham 
believed  separated  two  entirely  different  classes  of  matter.  Although 
at  the  present  time  the  differences  between  the  two  classes  do  not 
seem  so  great,  yet  the  same  division  is  found  useful  in  classification. 
By  colloids  Graham  indicated  those  substances  which  were  dissolved 
to  the  extent  of  showing  no  visible  particles  in  suspension,  but  which 
either  did  not  pass  through  diffusion  membranes  at  all,  or  did  so  very 
slowly  indeed,  as  compared  to  the  crystalloid  substances.  Under  cer- 
tain conditions  they  tended  to  assume  a  sticky,  glue-like  nature, 
hence  the  name.  (Many  substances  are  now  known  which  have  the 
chief  properties  of  the  colloids  and  are  therefore  classified  among 
them,  but  never  are  glue-hke,  e.  g.,  the  colloidal  metals,  so  that  the 
name  has  lost  some  of  its  original  significance.)  The  phj^sical  prop- 
erty which  Graham  particularly  noted  in  the  colloids,  besides  their 
non-difTusibility  was  the  tendency  to  assume  various  states  of  solidity. 

2°  For  further  consideration  of  the  subject  of  osmotic  pressure  in  these  rela- 
tions, see:  Livingston,  "The  Role  of  Diffusion  and  Osmotic  Pressure  in  Plants," 
University  of  Chicago  Press,  Chicago,  1903;  Czapek,  "Biochemie  der  Pflanzen," 
Jena.     Also,  Spiro,  Pauli  and  Hober,  all  previously  cited. 

^'  For  full  discussions  of  the  nature  of  colloids  see:  Hober,  " Physikalische 
Chemie  der  Zelle,"  Leipzig,  1914;  Pauli,  Ergebnisse  der  Physiologie,  1907  (6), 
105;  Bechhold,  "Colloids  in  Biology  and  Medicine,"  translated  by  J.  G.  M. 
Bullowa,  1919;  Wo.  Ostwald,  "Grundriss  der  Kolloidchemie,"  and  "Theoretical 
and  Applied  Colloid  Chemistry,"  both  translated  by  M.  H.  Fischer.  A  go9d 
brief  discussion  of  colloids  is  given  by  Young  in  Zinsser's  "Infection  and  Resis- 
tance." 


32  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

Not  only  can  they  be  in  solution,  when  he  called  them  "sols"  (when 
the  solvent  was  water,  "  hydrosols  ") ,  but  they  can  become  quite  firfn 
although  containing  much  water  (then  called  "gels"  or  "hydrogels"). 
The  gels  may  assume  a  firm,  coagulated  condition,  the  so-called  "pec- 
tous"  state,  which  state  is  permanent  in  that  the  gel  form  cannot  be 
reobtained  from  the  pectous  modification.  Finally  the  colloid  can  be 
in  a  dry,  solid  state,  quite  free  from  water,  and  then  not  a  gel  at  all. 

Included  in  the  great  class  of  colloids  are  all  forms  of  proteins, 
and  also  gums,  starch,  dextrin,  glycogen,  tannin,  probably  the  en- 
zymes, and  also  the  greater  number  of  organic  dyes;  also  there  are  in- 
organic colloids,  such  as  silicic  acid,  arsenic  sulphide,  hydrated  oxide 
of  iron,  and  many  other  similar  compounds,  besides  the  elements 
themselves,  especially  the  noble  metals,  which  may  exist  in  colloidal 
form.  It  will  be  seen  at  once  that  the  chief  constituents  of  the  cells, 
in  fact  nearly  all  the  primary  constituents  except  the  inorganic  salts, 
are  organic  colloids,  and  therefore  the  properties  of  the  cells  are  largely 
dependent  upon  the  properties  of  the  colloids. 

In  considering  the  characteristics  of  the  colloids  we  at  once  meet 
the  question — What  distinguishes  the  colloids  from  the  crystalloids, 
on  the  one  side,  and  from  suspensions  or  emulsions  on  the  other? 
The  sum  and  substance  of  our  present  conception  of  the  natiu-e  of 
colloidal  solution  may  be  brieflj''  sunmiarized  as  follows: 

It  is  possible  for  solid  substances  to  be  so  divided  among  the  par- 
ticles of  a  solvent  that  they  remain  permanentlj^  in  this  condition,  • 
neither  aggregating  into  masses  nor  separating  out  through  the  action 
of  gravity.  With  some  substances,  as  sugar,  for  example,  the  solid 
seems  to  divide  up  into  its  molecular  form,  each  molecule  being  free  from 
all  others  of  its  kind  except  during  occasional  contacts.  Some  other 
substances,  as  salt,  go  still  further,  and  the  molecule  cUvides  into  two 
or  more  parts,  which  have  different  electric  charges  {ionization).  The 
first  of  these  classes  of  substances  forms  a  solution  which  contains  no 
particles  visible  by  any  known  means,  does  not  contain  particles  large 
enough  to  reflect  light  imioingiiig  upon  them,  exerts  a  large  osmotic 
pressure,  but  does  not  conduct  electricity.  The  other,  in  which 
ionization  has  occurred,  differs  solely  in  its  capacity  to  conduct  elec- 
tricity readily.  Both  are  true  solutions  of  crystalloids;  the  one  which 
does  not  ionize  is  a  n.on-electroh/tc;  the  other,  by  virtue  of  its  ionization, 
is  an  electrolyte,  the  ions  carrj'ing  electric  charges  through  the  solution. 

At  the  other  end  of  the  scale  we  have  substances  which  are  quite 
insoluble  when  in  masses,  but  which,  when  very  finely  divided  by  me- 
chanical means,  can  l)e  sus])ended  and  uniformly  distributiul  through 
a  fluid  without  having  any  maiked  tendency  to  aggregate  or  settles 
out.  Such  suspensions  or  emulsions  contain  particles  visible  under 
the  microscope,  usually  appe;ii  Imhid,  refract  light,  are  non-diffusible, 
exert  no  osmotic  pressure,  and  do  not  transmit  electricity.  .Such 
mixtur(!s  are  obviously  veiy  dilTca'cnt  from  the  true  solutions  above 


COLLOIDS  33 

described.  Between  these  two  extremes  stand  the  colloids,  which  vary- 
in  tlieir  properties  so  that  they  approach  sometimes  the  suspensions 
(e.  g.,  lecithin,  or  coagulated  egg-albumin  in  colloidal  suspension), 
and  sometimes  more  nearly  the  true  solutions  (e.  g.,  dextrin).  No 
sharp  boundaries  can  be  drawn  between  any  of  the  members  of  the 
series.  Indeed,  one  substance  may  present  all  the  different  stages 
under  different  conditions,  some  agreeing  with  the  properties  of  the 
typical  suspensions,  and  some  with  the  properties  of  the  true  solutions. 
The  colloids  stand  in  an  intermediary  position,  differing  quantitatively 
in  one  way  or  another  from  the  true  solutions,  but  yet  approaching 
them  closely  and  sometimes  almost  indistinguishably  resembling  them. 
For  the  most  part,  however,  they  show  characteristics  decided  enough 
to  entitle  them  to  separate  classification,  and  to  make  any  confusion 
with  the  crystalloids  impossible. 

The  Characteristics  of  Colloids. — The  chief  properties  of  the 
colloids 'are,  then,  as  follows:  -  s^ 

Amorphous  Form. — This,  like  almost  all  other  "colloidal  properties,"  is  not 
absolute,  for  in  egg-albumin,  hemoglobin,  and  various  globuUns  we  have  proteins 
which  in  every  respect  are  typical  colloids,  yet  they  form  crystals  readily  and 
abundantly.  Oxyhemoglobin,  the  molecular  weight  of  which  is  calculated  at 
about  14,000.  exhibits  Tyndall's  phenomenon  and  will  not  pass  through  a  very  fine 
porcelain  filter,  and  therefore  resembles  the  colloids  decidedly,  yet  it  forms  beautiful 
crystals.  The  very  fact  that  crystals  are  formed,  Spiro  points  out,  is  proof  that 
when  in  solution  the  individual  molecules  must  have  been  free  and  separate,  for 
other\\dse  they  could  scarcely  unite  in  the  definite  spatial  relations  necessary  to 
produce  crystalline  forms.  With  these  few  exceptions,  however,  the  colloids  do 
not  present  any  typical  structure,  and  are  not  crystalUne  under  anj^  visible  condi- 
tion. But  when  they  are  made  insoluble  by  chemical  means  they  may,  under 
certain  conditions,  produce  rather  characteristic  non-crystalline  structures,  a 
matter  that  ^\•ill  be  discussed  in  a  subsequent  paragraph. 

Solubility. — Although  we  speak  of  "colloidal  solutions,"  this  terra  does  not 
commit  us  to  the  theory  of  the  identity  of  the  solution  of  colloids  ^^^th  that  of 
crystalloids.  We  have  above  stated  wliat  seems  to  be  a  fair  view  of  the  matter 
as  shown  by  many  methods  of  experimentation.  Most  colloids  seem  to  be,  in 
fact,  suspensions  of  masses  of  molecules,  or  perhaps  of  very  large  single  molecules, 
and  a  true  solution  is  Likewise  a  suspension  of  single  molecules  or  of  ions.  When 
the  aggregations  of  molecules  are  sufficiently  large,  we  have  an  ordinarj^  sus- 
pension; but  a  single  protein  molecule  is  as  large  as  a  very  great  number  of  mole- 
cules of  such  substances  as  sugar  (crystalloid);  or  tannin,  C14H10O9  (colloid);  or 
calcium  carbonate  (insoluble,  suspension);  and  it  would  be  strange  if  a  true 
solution  of  a  protein  did  not  behave  in  many  particulars  Uke  a  suspension  of  mo- 
lecular aggregates  of  dimensions  similar  to  the  dimensions  of  protein  molecules. 
Nearly  all  colloidal  solutions  show  Tj^ndall's  phenomenon,  \yhich  demonstrates 
the  existence  of  particles  in  saspension  large  enough  to  reflect  Ught  from  their  sur- 
faces." Most  of  the  colloids  are  held  back  by  very  fine  filters  to  a  greater  or  less 
degree;  some  are  almost  entirely  retained  by  a  hardened  paper  filter,  while  others 
pass  through  the  finest-pored  clay  filters.'  Furthermore,  the  metallic  colloids, 
such  as  those  of  platinum,  gold,  and  silver,  are  unquestionably  suspensions  of 
finely  di\dded  particles  of  metal,  yet  they  exhibit  all  the  typical  phenomena  of 
colloids,  passing  through  many  sorts  of  filters,  and  even  inducing  the  same  hydro- 
lytic  changes  as  many  enzymes. 

"  It  is  highly  probable,  however,  that  Tyndall's  phenomenon  when  exhibited 
by  true  colloidal  solutions  (e.  g.,  soluble  proteins),  depends  on  the  presence  of  aggre- 
gates and  not  properly  on  the  dissolved  colloids.  (See  McClendon  and  Prender- 
gast.  Jour.  Biol.  Chem.,  1919  (38),  549.) 

3 


34  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

It  must  also  be  mentioned  that  the  solvent  is  probably  an  important  factor 
in  determining  the  colloidal  or  non-colloidal  nature  of  a  substance;  e.  g.,  soaps  form 
true  solutions  in  alcohol  and  colloidal  solutions  in  water;  gelatin  forms  colloidal 
solutions  in  water  but  not  in  ether,  whereas  rubber  forms  colloidal  solutions  in 
ether  but  not  in  water. 

Closely  related  to  solubility  is  the  phenomenon  of  imbibition,  which  may  be 
defined  as  the  taking  up  of  a  fluid  by  a  solid  body  wathout  chemical  change.  Not 
all  colloids  possess  this  property,  but  it  is  shown  by  most  of  the  organic  colloids, 
particularly  the  proteins.  Fick  distinguishes  capillary,  osmotic,  and  molecular 
imbibition,  the  latter  of  which  is  the  form  exhibited  by  colloids,  and  it  occurs  in- 
dependent of  the  existence  of  pores  or  other  preformed  spaces  in  the  imbibing  body. 
The  imbibition  of  water  by  colloids  is  more  than  a  simple  mechanical  process,  for 
it  is  accompanied  by  a  contraction  in  the  total  volume  of  solid  and  water,  and  by 
the  evolution  of  heat.  The  forces  developed  are  far  greater  than  those  of  osmotic 
pressure;  e.  g.,  to  prevent  imbil>ition  of  water  by  starcli  requires  a  pressure  of  over 
2500  atmospheres.  On  the  otlier  hand,  the  physical  properties  of  an  aqueous 
colloidal  solution  show  that  the  colloid  is  not  chemically  combined  in  the  form  of  a 
hydrate.  To  describe  this  peculiar  relation  Hofmeister  and  Oswald  recommend 
the  term  "mechanical  affinity."  Hardy  has  shown  that  water  held  in  a  gelatin 
jelly  cannot  be  removed  by  great  pressures  (400  pounds  to  the  square  inch),  but 
after  the  nature  of  the  jelly  is  so  changed  by  formalin  that  it  is  no  longer  liquefiable 
by  heat,  the  water  can  be  easily  expressed  from  the  loose  meshwork  that  is  formed. 
It  would  seem  from  this  that  the  imbibition  and  retention  of  water  by  colloids  may 
be  closely  related  to  surface  phenomena.  Hofmeister  has  shown  that  organized 
animal  tissues  obey  the  same  laws  of  imbibition  as  do  simple  gelatin  plates,  and 
probably  this  phenomenon  of  colloids  is  very  important  in  physiological  and  patho- 
logical processes. 

Non-diffusibility. — The  lack  of  power  to  pass  through  animal  and  parchment 
membranes,  which  was  Graham's  starting-point  in  the  study  of  colloids,  is  also 
only  a  relative  condition.  This  is  shown  by  the  following  figures,  giving  the  rela- 
tive time  required  by  the  same  amount  of  different  substances  to  pass  through  a 
certain  diffusion  membrane: 

Sodium  chloride 2 .  33 

Sugar 7.00 

Magnesium  sulphate 7 .  00 

Protein 49.00 

Caramel 98.00 

This  difference  of  time  is  so  great,  however,  as  to  permit  of  separation  of  salts 
from  proteins,  etc.,  by  dialysis,  a  process  in  constant  use.  Primarily  the  ability 
to  diffuse  through  a  given  membrane  requires  that  the  diffusing  substance  be 
soluble  in  the  membrane.  Diffusion  menil)ranes  are  always  composed  of  colloids, 
e.  g.,  animal  bladders,  or  parchment,  whicli  is  a  colloidal  cellulose.  Crystalloids 
are  generally  solul>le  in  colloids,  while  colloids  are  little  or  not  at  all  soluble  in 
other  colloids,  and  hence  do  not  dilTuse  through  one  another  readily  and  permeate 
diffusion  membranes  very  slowly.  For  example,  if  a  stick  of  agar  jelly  be  jilaced 
in  a  solution  of  ammoniated  copper  sulphate  (a  crystalloid),  and  another  be  placed 
in  a  solution  of  Prussian  blue  (a  colloid),  it  will  be  found  that  the  copper  solution 
penetrates  the  agar  completely  before  the  colloidal  solution  of  Pru.ssian  blue  has 
penetrated  it  at  all.  This  property  is  of  great  importance,  undoubtedly,  in  keep- 
ing different  colloidal  constituents  of  the  cell  in  given  localities  witliin  its  proto- 
plasm, c.  g.,  tl)e  colloidal  glycogen  remains  wliere  it  is  formcil  in  tlie  cytoplasm, 
unaljle  to  escape  from  tlie  cell,  whereas  the  crystalloidal  sugar  from  which  it  is 
formed  and  into  which  it  is  converted,  diffuses  raiiitlly  into  or  out  of  the  cell. 
The  osmotic  pressure  of  tiie  colloids  is  extremely  small.  The  closely  related 
phencjinena  of  dijliision,  (U'prc.ss'ion  of  freezing-point,  and  ehrnlion  of  boiling-point, 
are  also  exhibited  by  colloids  to  l)ut  an  extremely  slight  degree.  For  example,  in 
one  experiment,  tlie  di.s.solving  of  from  14  per  cent,  to  44  ]K'r  cent,  of  egg-albumin 
in  water  lowered  the  freezing-point  but  0.02°  to  0.00°;  and  some  other  colloids 
have  even  less  effect.  The  results  of  the  latest  and  best  experiments  seem  to  in- 
dicate tiiat  the  trifling  ericcts  of  colloids  upon  osmotic  pressure  and  upon  freezing- 
and  l)oiling-i)oin1s  oiiserved  in  colloidal  solulions  are  due  to  the  colloids  tliemselves, 
ratlier   than   to  included  inii)urities,   although  it  may  possibly  be  th.at  some  oi 


COLLOIDS  35 

these  effocts  are  due  to  the  high  surfiice  tension  and  cohesion  afTnity  of  the  rolloids. 
In  all  cellular  processes  accompanied  by  manifestations  of  osmotic  pressure  or 
difTtision,  liowcner,  the  crystalloids  may  be  considered  as  almost  entirely  responsible. 
Electrical  Phenomena. — As  colloids  do  not  separate  freely  into  ions  when  di.s- 
solved,  they  do  not  con(hict  electricity  apprecial)ly.  However,  when  an  electric 
current  is  passed  throunh  water  containing  colloids  in  sf)lution,  the  colloidal  par- 
ticles tend  to  pass  to  one  ])ole  or  the  other.  Most  colloids  move  toward  the  anode. 
This  phenomenon,  cdln phoresis^,  is  also  generally  exhibited  by  suspensions,  and 
hence  in  this  particiUar  the  colloids  resemble  suspensions  rather  than  solutions. 
Ilelmholtz  has  explained  the  movement  of  the  suspended  particles  as  due  to  the 
accumulation  of  electrical  charges  upon  the  surfaces  of  two  heterogeneous  media 
when  in  contact.  The  nature  of  the  charge  depends  upon  both  the  suspended 
substance  and  the  fluid;  e.  g.,  sulphur  or  graphite  particles  siispended  in  water 
assume  a  negative  charge  and  move  toward  the  anode,  but  when  suspended  in  oil 
of  t\n-pentinc  they  become  positively  charged  and  move  toward  the  cathode. 
\N'ater  has  such  a  high  dielectric  co7istant  that  most  substances  suspended  in  water 
become  negatively  charged  as  compared  with  the  water,  and  move  toward  the 
positive  pole  or  anode. 

Hardy  has  observed  that  colloidal  solutions  of  coagulated  proteins  move  toward 
the  anode  when  in  alkaline  solution,  and  toward  the  cathode  when  in  acid  solu- 
tion.-'-' This  peculiar  i)roperty  of  proteins  suggests  that  perhaps  simple  surface 
phenomena  do  not  suffice  to  account  for  the  electrification  of  all  colloid  particles. 
Knowing  the  peculiar  amphoteric  character  of  proteins, which  is  probably  due  to 
the  presence  of  both  NHo  and  COOH  groups  in  the  molecule,  we  can  readily  under- 
stand that  in  an  acid  solution  the  NH2  radicles  are  combined  with  the  acid,leaving 
the  COOH  radicles  free.  The  molecule  would  then  have  acid  properties,  and  could 
dissociate  into  an  acid  H  ion  and  a  basic  or  electrically  positive  colloid  ion.  The 
colloid  ion  would  then  go  toward  the  negative  pole  slowly,  because  of  its  great 
size.  When  a  suitable  concentration  of  both  ions  is  produced  the  proteins  will 
move  towards  both  poles,  this  concentration  being,  in  the  case  of  serum  albumin, 
H  =  10~^  (Michaelis).  Living  protoplasm  behaves  in  most  instances,  as  if  the 
proteins  were  acids  bound  to  inorganic  cations  (Robertson),  and  is  usually  stimu- 
lated at  the  cathode  on  the  "make"  of  the  current.  It  is  permeable  to  ions,  and 
the  vitality  of  a  tissue  is  so  dependent  on  the  maintenance  of  normal  permeability 
that  the  permeability  may  be  employed  as  a  sensitive  and  reliable  indicator  of 
its  vitality  (Osterhout-^).  This  maj^  be  done  by  determining  the  electrical  resis- 
tance of  the  cells,  which  is  lowered  by  anything  that  lowers  their  vitality. 

Surface  tension,-^  which  may  be  described  as  the  force  ivilh  which  a  fluid  is 
striving  to  reduce  its  free  surface  to  a  minimum,  is  highly  exhibited  by  colloids  as 
compared  with  crystalloids.  The  formation  of  emulsions  and  the  spreading  out 
of  oil  upon  the  surface  of  water  depend  upon  surface  tension.  Ameboid  movement 
may  be  attributed  to  changes  in  surface  tension,  as  also  may  phagocytosis.  (The 
relation  of  surface  tension  to  these  processes  will  be  considered  under  the  subject 
of  Inflammation.) 

The  effect  of  colloids  upon  chemical  processes  going  on  within 
their  solutions  or  gels  is  surprisingly  small.  Salts  in  solution  in  a 
thick  gel  of  agar  or  gelatin  will  diffuse  almost  as  rapidly  as  in  water; 
they  will  also  ionize  as  rapidly  as  in  watery  solutions,  and  chemical 
reactions  occur  with  nearly  the  same  speed  and  completeness  as  if  the 
colloids  were  absent.  Furthermore  it  makes  little  difference  whether 
these  processes  are  measured  in  a  colloidal  solution  that  is  liquid,  or 
after  it  has  set  in  the  gel  form.  These  facts  merely  indicate  that  the 
colloids  do  not  greatly  impede  the  movements  of  molecules  or  ions  in 

"According  to  Field  and  Teague  (Jom-.  Exper.  Med.,  1907  (9),  222),  native 
proteins  in  serum  move  towards  the  cathode,  no  matter  what  the  reaction. 

-'  Science,  1914  (40),  488. 

^^  See  article  on  "Sm-face  Tension  and  Vital  Phenomena,"  by  Macallum,  Ergeb- 
nissed.  Physiol.,  1911  (11),  G02. 


36  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

solutions.  On  the  other  hand,  as  before  mentioned,  colloids  diffuse 
very  slowly  into  each  other.  Hence,  in  the  cell  the  colloids  are  quite 
fixed  in  their  positions,  whereas  the  crystalloids  may  wander  about 
freely,  and  tliis  arrangement  is  certainly  of  great  importance  in  bio- 
logic processes.  Pauli  suggests  the  probability  that  the  fixation  of  the 
colloid  causes  the  cell  to  have  different  properties  in  different  parts, 
and  so  various  reactions  may  occur  independently  in  different  areas 
of  the  cytoplasm.  The  possibility  of  the  correctness  of  this  view  is 
increased  when  we  consider  that  the  enzymes  are  colloids,  for  there  is 
much  evidence  to  show  that  thej^  are  distributed  in  just  such  an  uneven 
manner  within  the  cells. 

Although  colloids  permit  the  passage  of  dissolved  crystalloids 
through  them,  they  greatly  interfere  with  the  movement  of  larger 
particles.  This  property  accounts  for  the  ability  of  colloids  to  hold 
many  insoluble  substances  in  such  extremely  fine  suspensions  that 
they  seem  superficially  to  be  in  true  solution.  If,  for  example,  sodium 
phosphate  is  added  to  a  solution  of  casein  in  lime-water,  the  calcium 
phosphate  formed  does  not  precipitate.  It  is  not  in  solution,  how- 
ever, but  rather  exists  as  a  suspension  of  ver}^  finely  divided  particles 
of  the  salt  which  the  colloid  keeps  from  aggregating  into  particles 
large  enough  to  be  visible  or  to  overcome  the  viscosity  of  the  fluid 
and  sink  to  the  bottom.  Probably  in  this  way  many  substances,  in- 
cluding calcium  salts,  are  carried  in  the  blood,  held  in  permanent 
suspension  by  the  proteins.  Substances  thus  finely  chvided  w^ill  have 
extremely  large  surface  area  for  reactions,  and,  therefore,  will  undoubt- 
edly undergo  changes  with  considerable  rapidity  and  facility,  although 
not  in  solution. 

Precipitation  and  Coagulation  of  Colloids. — Because  of  the 
slender  margin  by  which  the  colloids  are  separated  from  the  suspen- 
sions, their  persistence  in  solution  is  generally  in  a  precarious  con- 
dition. Relatively  slight  changes  suffice  to  throw  the  colloids  out  of 
solution,  and  when  once  precipitated,  they  are  often  incapable  of 
again  dissolving  in  the  same  solvent.  Solutions  of  albumin  may  under- 
go spontaneous  coagulation  on  standing  for  some  time,  and  agitation 
rapidly  produces  the  same  effect  in  many  protein  solutions.  Some 
inorganic  colloids  are  as  readily  coagulated  as  the  proteins.  A  com- 
paratively small  rise  in  temperature,  less  than  to  50°  C.  with  some 
proteins,  renders  the  protein  perfectly  insoluble.  Furthermore,  we 
have  coagulation  of  protein  solutions  by  enzyme  action.  The  inor- 
ganic "colloidal  suspensions"  may  be  precipitated  by  the  addition  of 
very  small  quantities  of  electrolytes.  Colloidal  solutions  of  the  tj'pe 
of  the  proteins  are  not  so  readily  jireciintaled  by  most  clectrolj'tes, 
but  if  to  the  solution  large  quantities  of  crystalloids  are  added,  the 
protein  molecules  are  practically  crowded  out  of  solution,  as  in  the 
"salting-out"  process  used  in  separating  proteins  by  ammonium  sul- 
phate and  other  salts.     The  effect  of  heat  upon  different  colloids  is 


COLLOIDS  37 

peculiar,  in  that  sonic  varieties,  as  silicic  acid,  aluininiuni  hydrate,  and 
many  proteins  are  rendered  so  insoluble  that  they  cannot  again  be 
dissolved  in  any  fluid  without  first  being  modified  in  some  way;  where- 
as colloids  of  the  type  of  gelatin  and  agar  are  made  more  soluble  by 
heat.  The  change  of  colloids  into  insoluble  forms,  the  "pedous" 
condition  of  Graham,  requires  the  presence  of  water,  for  the  dry  col- 
loids may  be  heated  to  relativelj''  high  temperatures  without  losing 
their  solubility.  On  the  other  hand,  dehydration  of  colloids  while  in 
solution  will  result  in  their  precipitation  and  coagulation,  as  occurs  in 
protein  solutions  when  alcohol  is  added. 

If  solutions  of  two  oppositely  charged  colloids  are  brought  together 
they  may  precipitate,  but  if  either  is  present  in  excess  the  precipita- 
tion may  be  incomplete,  or  even  completely  absent.  This  inhibition 
of  precipitation  is  of  particular  interest  because  it  so  closely  resembles 
the  phenomenon  observed  in  the  precipitin  reaction,  whereby  an 
excess  of  the  antigenic  protein  will  prevent  precipitation.  Also  cer- 
tain colloids  will  prevent  the  precipitation  of  other  colloids  by  elec- 
trolytes, which  fact  is  the  basis  of  the  Lange  reaction  of  spinal  fluid 
with  colloidal  gold. 

Colloids  are  precipitated  by  many  electrolytes,  apparently  through 
the  formation  of  true  ion  compounds,  one  or  both  of  the  ions  of  the 
electrolytes  uniting  with  the  colloid  ion;  although  some  writers,  as 
Spiro,  believe  that  the  combination  is  merely  an  additive  one  between 
entire  molecules.  Mathews^^  has  advanced  the  theory  that  the  solu- 
tion tension  of  the  ions  is  an  important  factor  in  determining  the  pre- 
cipitation of  colloids  by  electrolytes.  In  general,  precipitation  of 
colloids  results  from  the  reduction  of  the  surface  in  proportion  to  the 
mass,  because  of  an  aggregation  of  the  particles;  this  may  be  brought 
about  by  changing  the  surface  electrical  conditions,  by  uniting  the 
molecules  chemically,  or  by  reducing  the  amount  of  the  solvent. 

The  Structure  of  Colloids  and  of  Protoplasm. ^^ — Two  very 
different  sorts  of  substances  are  usually  included  under  the  term  colloid, 
because  they  show  the  essential  features  of  colloids  in  most  respects; 
but  as  in  many  other  respects  they  are  quite  unlike  each  other,  it  may 
be  well  to  distinguish  between  them  in  some  way.  As  a  type  of  one 
class  we  may  take  gelatin;  of  the  other,  such  a  substance  as  colloidal 
arsenious  sulphide.  Gelatin  solutions  form  gels  upon  cooling  or  evap- 
oration, and  redissolve  when  heated  or  when  more  solvent  is  added. 
Arsenious  sulphide  does  not  form  gels  upon  cooling,  and  when  solidified 
in  any  way,  does  not  redissolve.  In  addition,  the  gelatin  type  is  very 
viscous,  and  is  not  coagulated  by  the  presence  of  salts  unless  these  are 
added  in  large  amounts;  while  the  other  type  does  not  render  the  fluid 
in  which  it  is  dissolved  appreciably  more  viscid,  and  it  forms  a  precipi- 
tate immediately  if  minute  amounts  of  electrolytes  are  introduced. 

^*  American  Journal  of  Physiology,  1905  (14),  203. 

"  Review  by  Harper,  Amer.  Jour.  Botany,  1919  (6),  273. 


38  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

As  the  former  type  resembles  in  many  details  the  true  solutions,  while 
the  latter  approaches  more  closely  to  the  suspensions,  it  has  been 
proposed  to  distinguish  them  by  the  terms  "colloidal  solution"  and 
"colloidal  suspension."-'^  Of  the  two  types,  the  colloidal  solutions 
are  by  far  the  more  important  in  biological  considerations,  since  the 
colloidal  suspensions  are  usually  prepared  artificially  and  seldom  occur 
in  nature,  e.  g.,  Bredig's  colloidal  suspensions  of  the  noble  metals. 

The  colloidal  solutions  of  proteins  are  of  two  tj^pes — one,  such  as 
albumin,  forms  a  coagulum  when  heated,  which  under  ordinary 
conditions  is  not  reversible;  that  is,  it  does  not  again  go  into  solution. 
Gelatin,  however,  becomes  more  fluid  when  heated,  and  when  cooled 
it  forms  a  gel  which  is  readily  reversible  to  the  soluble  form  under  the 
influence  of  heat.  Within  the  cell,  as  far  as  we  know,  occur  only  the 
first  type,  the  proteins  that  form  non-reversible  coagula. 

An  extensive  study  of  the  physical  structure  of  the  colloids  has 
been  made  by  Hardy. ^^  As  long  as  the  colloid  is  in  solution  it  is 
structureless,  although,  as  before  mentioned,  the  existence  of  free 
solid  particles  can  be  demonstrated  by  certain  optical  methods.  The 
solution  is  homogeneous,  and  although  perhaps  viscid,  still  it  is  a  typ- 
ical solution.  Such  solutions  can  become  solid,  either  by  the  effect  of 
temperature,  of  certain  chemical  fixing  agents,  or  physical  means. 
It  was  found  by  Hardy  that  in  undergoing  this  solidification  there  oc-' 
curs  a  separation  of  the  solid  from  the  liquid,  the  solid  particles 
adhering  to  form  a  framework  holding  the  liquid  within  its  interstices. 
Heat-reversible  gels  show  no  structure  until  they  are  made  irreversible 
by  hardening  agents,  etc.;  e.  g.,  a  jelly  of  gelatin  appears  structui'e'ess, 
but  when  treated  with  formalin  or  other  fixing  agent,  the  structural 
appearances  described  below  appear.  The  figures  formed  by  the 
framework  vary  according  to  the  nature  and  concentration  of  the 
colloid  and  of  the  solvent,  and  also  with  the  fixing  agent  used,  the 
temperature,  and  the  presence  or  absence  of  extraneous  substances. 
In  general,  however,  the  figures  obtained  in  the  solidification  of  pro- 
tein solutions  by  fixing  agents,  such  as  bichloride  of  mercury  or 
formalin,  bear  a  striking  resemblance  to  the  finer  structures  of  protoplasm 
as  described  by  cytologists.  There  is  produced  an  open  network 
structure  with  spherical  masses  at  the  nodal  points,  or  minute  vesicles 
hollowed  out  in  a  solid  mass,  or  a  honej'comb  appearance,  or,  when  the 
concentration  of  the  colloid  is  very  slight,  perhaps  there  is  only  a 
precipitation  of  fine  granules  of  protein  such  as  we  often  see  in  histo- 
logical preparations  of  edematous  cells  and  tissues.  All  these  forms 
seem  to  depend  chiefly  uj^on  the  concentration  of  the  colloid.  The 
important  fact  is  that  when  the  chemicals  ordinarily  used  as  fixatives 
of  cells  for  histological  purposes  act  upon  solutions  of  colloids  that 
are  perfectly  homogeneous,  they  produce  very  constant  and  charac- 

^*  Noyes,  American  Cliemical  Journal,  1905  (27),  85. 
2»  Journal  of  Physiology,  1899  (24),  158. 


CELL  STRUCTURE  39 

tcristic  formations  wliicli  recall  at  once  the  structures  found  in  the 
protoplasm  of  hardened  colls.  Moreover,  the  use  of  different  fixing 
agents,  such  as  osmic  acid,  formalin,  and  bichloride  of  mercury,  pro- 
duces just  the  same  differences  in  the  structure  of  colloidal  solutions 
that  they  produce  in  the  protoplasm  of  cells  hardened  by  tliom. 
Neither  are  the  appearances  seen  in  unfixed  specimens  reliable  indi- 
cations of  the  true  structure  of  the  living  protoplasm.  Granules  of 
secretion  may  disappear  after  or  during  the  death  of  the  cell  (e.  (j., 
glycogen)  or  they  may  swell  up  (e.  g.,  mucin  granules),  thus  giving 
the  aj^pcarance  of  a  network  or  honeycomb  which  is  then  incorrectly 
ascribed  to  the  protoplasm  itself.  Death  of  the  cells,  even  when  not 
produced  by  external  influences,  seems  to  be  accompanied  by  coagula- 
tion of  some  parts  of  the  cell  constituents,  and  hence  a  cell  examined 
in  anything  but  its  normal  living  condition,  an  extremely  difficult 
matter,  will  not  present  a  true  idea  of  how  it  appears  and  is  composed 
while  in  that  condition.  By  microdissection  with  the  Barber  pipette 
method  it  is  possible  to  study  the  properties  of  unaltered  living  cyto- 
plasm, and  Seifriz^"  concludes  from  his  studies  that  protoplasm  is 
a  homogeneous  structureless  solution,  probably  an  emulsion  hydrosol, 
i.  e.,  a  colloid  in  which  both  phases  are  liquid,  one  of  them,  the  disper- 
sion medium,  being  water.  Normal  cytoplasm  is  at  all  times  non- 
miscible  in  water,  but  readily  degenerates  into  a  condition  in  which  it 
is  miscible. 

If,  with  these  facts  in  mind,  we  consider  the  theories  of  morpholo- 
gists  as  to  the  finer  structure  of  the  cell  protoplasm  based  upon  stud- 
ies of  cells  fixed  in  various  hardening  agents,  it  becomes  evident  that 
the  possibility  that  the  "foam  structure"  advocated  by  Biitschli,  or 
the  "thread,"  "reticular,"  and  "pseudo-alveolar"  structures  of  Fro- 
mann,  Arnold,  Reinke,  and  others,  are  all  simply  the  effect  of  fixatives 
upon  colloid  solutions,  is  very  real.  The  objection  always  advanced 
to  these  theories  of  protoplasmic  structure,  namely,  that  the  structures 
described  were  at  least  in  part  artificial  productions,  not  present  in  the 
normal  living  cell,  and  variously  described  and  interpreted  by  differ- 
ent investigators,  because  each  worked  with  a  chfferent  hardening 
fluid  or  different  technic,  is  strongly  supported  by  these  observations 
upon  colloids.  This  matter  will  receive  further  consideration  in  the 
next  section. 

THE  STRUCTURE  OF  THE  CELL  IN  RELATION  TO  ITS  CHEMISTRY 

AND  PHYSICS" 

It  is  obviously  impossible  to  separate  nuclei,  nucleoh,  cytoplasm, 
and  cell  membranes  from  each  other  (except  with  sperm  heads)  and 
to  isolate  them  in  quantities  sufficient  for  analysis,  and  therefore  we 

=»  Biol.  Bull.,  1918  (34),  307. 

'^  Reviews  of  the  significance  of  cell  structure  for  pathology  are  given  by  Benda 
and  Ernst  in  Zentrlbl.  allg.  Path.,  1914,  Bd.  25,  Ergiinzungsheft. 


40  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

are  still  quite  uncertain  as  to  just  the  chemical  differences  that  exist 
between  them.  That  there  are  differences  is  certain,  and  by  means  of 
micro-chemical  reactions,  by  comparing  analyses  of  cells  in  which 
nucleus  or  cytoplasm  predominate,  and  by  studying  their  phj'sico- 
chemical  relations  to  one  another,  we  have  arrived  at  more  or  less 
tangible  ideas  on  the  question  of  the  relation  of  the  structural  elements 
of  the  cell  to  its  compositi  m. 

The  Nucleus  32 

Although  the  nucleus  presents  morphologically  a  sharp  isolation 
from  the  cytoplasm,  and  displays  equally  sharp  tinctorial  differences, 
it  is  probable  that  chemically  the  differences  between  nucleus  and  c^^to- 
plasm  are  quantitative  rather  than  qualitative.  The  characteristic 
affinity  of  certain  elements  of  the  nucleus  for  basic  stains  depends 
upon  the  presence  in  the  nucleus  of  nucleoproteins  in  large  proportion, 
and  to  a  limited  degree  nucleoproteins  are  characteristic  of  nuclei. 
Their  affinity  for  basic  dyes  depends  upon  the  nucleic  acid  radical. ^^ 
For  example,  the  heads  of  spermatozoa  contain  nucleic  acid  bound  to 
simple  proteins  in  such  a  way  that  it  readily  forms  a  salt  or  salt-like 
combination  with  basic  dyes,  and  so  the  sperm  heads  appear  intensely 
stained  by  alum-hematoxylin,  etc.  Ordinary  chromatin  threads  of 
nuclei  appear  to  contain  somewhat  more  firmly  bound  protein  in  their 
nucleoprotein  molecules,  and  hence  stain  less  intensely  than  do  the 
spermatozoa  heads,  except  when  in  karyokinesis,  when  the  chromatin 
nucleoprotein  seems  to  approach  that  of  the  spermatozoa  in  avidity 
for  basic  dyes.  We  also  have  nucleoproteins  with  the  nucleic  acid  so 
thoroughly  saturated  by  protein  that  they  do  not  stain  at  all  by  basic 
dyes,  and  these  seem  to  exist  principally  in  the  cytoplasm,  and  also  to 
form  the  ground-substance  of  the  nuclei,  occupying  the  spaces  between 
the  chromatin  particles  (this  achromatic  substance  of  the  nuclei  is  called 
linin  or  plastin  by  some  cytologists) .  Besides  the  chromatin  and  the 
nucleoli,  there  is  a  peculiar  chromatophile  substance,  suspended  in  the 
finer  part  of  the  nuclear  structure  in  the  same  manner  as  the  chro- 
matin itself  is  in  the  coarser  portions;  this  was  called  lanthanin  by 
Heidenhain,^''  and  is  probably  similar  to  the  substances  also  described 
as  parachromatin  and  paralinin.  Undoubtedly  the  other  forms  of  pro- 
teins found  in  the  cell,  such  as  globulin,  albumin,  and  nucleoalbumin, 
exist  both  in  the  nucleoplasm  and  in  the  cytoplasm,  the  essential  dif- 
ference being  that  the  proportion  of  nucleoprotein  is  nuich  greater  in 
the  nucleus.     As  nucleoproteins  arc  little  alTectcd  bj^  peptic  digestion, 

32  Earlier  literature  bv  Albrecht,  "Pathologic  der  Zelle,"  Lubarsch-Ostertag, 
Ergeb.  der.  allg.  Pathol.,  1899  (6),  1900:  see  also  Kossel,  Miinch.  mcd.  Woch.,  1911 
(58),  05. 

-■'■'  Herwerdon  (Arch.  Zellforsch.,  1913  (19),  431)  found  that  the  basophilic 
gnmuUvs  are  disiutcgrated  specifically  by  nuclease,  supporting  the  view  that  they 
are  nucleic  acid  compounds. 

■>*  Festschr.  f.  Kollikcr,  1892,  p.  128. 


CELL  STRUCTURE  41 

it  is  possible  to  isolate  nuclear  elements,  especially  the  chromatin,  for 
analytic  purposes,  and  it  has  been  demonstrated  by  this  means  also 
that  nuclein  is  the  chief  constituent  of  the  staining  elements.  The 
distribution  in  the  nucleus  of  the  other  primary  constituents  of  the 
cytoplasm,  such  as  lecithin,  cholesterol,  and  inorganic  salts  has  not 
yet  been  worked  out,  except  that  Macalluni"''^  found  that  nuclei  contain 
no  chloride,  as  indicated  by  their  not  staining  with  silver  nitrate,  and 
also  no  potassium, ^^  but  the  chromatin  contains  firmly  bound  iron. 

Nucleoli,  which  not  all  varieties  of  nuclei  possess,  differ  from  the'other  nuclear 
structures  in  having  an  affinity  for  acid  rather  than  for  basic  dyes,"  at  least  in  fixed 
tissues.  Their  chemical  composition  lias  not  been  ascertained.  Zacharias  con- 
siders the  nucleoli  as  composed  of  nuclein  well  saturated  with  protein,  because  of 
its  staining  reactions  and  its  relative  insolubility  in  alkalies,  and  classes  it  with 
plastin  or  linin,  which  forms  the  achromatic  part  of  the  nucleus  and  is  also  present 
in  the  cytoplasm.  Macallum'*  found  that  they  reacted  for  organic  phosphorus 
microchemically,  but  less  strongly  than  did  chromatin  fibers. 

The- nuclear  membrane  is  an  imcertain  structure,  at  times  dense  and  staining 
as  if  formed  of  a  layer  of  chromatin,  in  other  cells  staining  like  the  cytoplasm  with 
which  it  seems  to  be  continuous,  in  most  cells  disappearing  during  karyokinesis, 
and  in  some  protozoa  being  entirely  absent.  Naturally  the  composition  of  the 
nuclear  membrane  is  unknown,  but  it  is  probable  that  it  acts  as  a  diffusion  mern- 
brane  of  partially  semipermeable  character,  maintaining  different  conditions  in 
nucleus  and  cytoplasm. 

Functionall}^  the  nucleus  is  the  essential  element  of  the  cell;  an 
isolated  nucleus  with  but  a  minimum  of  cytoplasm  may  be  able  to  de- 
velop new  cytoplasm,  but  isolated  cytoplasm  soon  disintegrates,  al- 
though it  may  manifest  Hfe  for  some  time  by  movement  and  chemical 
activities.  It  has  been  frequently  suggested  that  the  nucleus  controls 
oxidative  processes,  and  there  is  some  microchemical  evidence  for 
this.^^  Lynch-^"  calls  attention  to  the  improbability  that  the  part  of 
the  cell  most  removed  from  the  oxygen  should  be  the  organ  of  oxida- 
tion, and  finds  evidence  that  the  function  of  the  nucleus  is  that  of 
organic  synthesis.  An  enucleated  cell  may  move,  respire,  digest, 
respond  to  stimuli  and  exhibit  any  activity  which  is  dependent  solely 
upon  catabolic  or  destructive  processes  of  protoplasm.  The  group  of 
phenomena  which  it  never  shows  are  those  of  growth,  regeneration  and 
division,  i.  e.,  those  depending  on  synthetic  activities. 

It  should  be  mentioned  that  certain  cells,  such  as  bacteria  and  algse, 
'seem  to  have  no  true  nuclei,  but  Macallum^^  found  that  the  forms  he 
examined  gave  reactions  for  phosphorus  and  iron  in  a  similar  way 
to  the  nucleoproteins  of  a  nucleus,  suggesting  that  in  such  cells  the 
nuclear  elements  are  diffused  through  the  cell  rather  than  differen- 
tiated.    To*  quote   Wilson:  "The   term    'nucleus'    and   'cell   body' 

35  Proceedings  of  the  Roval  Society,  1905  (76),  217. 

se  Jour,  of  Physiol.,  1905  (32),  95. 

3^  Nucleoli  of  nerve-cells  are  an  exception,  being  basophilic. 

=58  Proc.  of  the  Roval  Societv,  1898  (63),  467. 

33  See  Osterhaut,  Science,  1917  (46),  367. 

«  Amer.  Jour.  Physiol.,  1919  (48),  258. 

*^  "Studies  from  the  University  of  Toronto,"  1900. 


42  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

should  probably  be  regarded  as  only  topographical  expressions,  de- 
noting two  differentiated  areas  in  a  common  structural  basis." 

Because  of  the  relative  acidity  of  the  nuclei  they  are  electrically 
negative  to  the  cytoplasm,  particularly  when  in  karyokinesis,  and  the 
chromatic  elements  of  the  nucleus  can  be  shown  to  carrj'"  a  negative 
electric  charge."^-  Sperm-heads  in  isotonic  cane-sugar  solution  move 
rapidly — ^2000  microns  a  minute — ^toward  the  anode,  when  a  current 
is  passed  through  the  solution;  and  leucocytes  also  go  toward  the 
anode  under  the  same  conditions,  the  rate  depending  upon  the  pro- 
portion of  nucleoplasm  and  cytoplasm,  large  leucocytes  sometimes 
even  going  slowly  toward  the  cathode.  The  SertoH  cells  of  the  testi- 
cle, which  have  a  round  mass  of  cytoplasm  with  a  number  of  minia- 
ture spermatozoa  heads  at  one  side,  orient  themselves  in  the  current 
so  that  the  side  or  end  containing  the  spermatozoa  drags  the  mass 
of  cytoplasm  toward  the  positive  pole. 

The  Cytoplasm 

The  cytoplasm,  as  before  mentioned,  contains  all  the  primary 
cellular  constituents,  and  also  such  secondary  constituents  as  the  par- 
ticular cell  possesses.  Nucleoproteins  are  undoubtedly  present  in 
unknown  proportions,  but  with  the  nucleic  acid  well  saturated  by 
proteins,  and  perhaps  also  to  a  large  extent  combined  with  carbohy- 
drates to  form  the  glyconucleoproteins.  Sometimes  the  nucleoproteins 
of  the  cytoplasm  may  be  partly  of  the  unsaturated  class,  and  show 
an  affinity  for  basic  stains,  as  in  the  case  of  the  Nissl  bodies  of  the 
nerve-cells,  the  basophilic  granules  of  mast  cells,^^  and  perhaps  also 
the  cytoplasm  of  plasma  cells.  The  great  question  concerning  the 
cytoplasm  is  its  structure — whether  homogeneous,  alveolar,  areolar, 
fibrillar,  foam-like,  or  granular.  On  a  previous  page  have  been  men- 
tioned the  experiments  of  Hardy,  which  show  that  homogeneous  solu- 
tions of  protein,  when  fixed  by  the  same  reagents  as  are  used  in 
the  customary  fixation  of  histological  materials,  may  show  quite 
the  same  microscopical  structures  as  are  shown  by  the  cytoplasm 
of  cells.  Network,  foam,  and  alveolar  structures  are  produced  in 
albumin  and  gelatin  solutions  when  they  are  hardened  by  bichloride 
of  mercury,  osmic  acid,  formalin,  etc.,  and  the  same  characteristic 
differences  that  are  produced  in  cells  by  these  different  reagents  are 
likewise  produced  in  the  hardened  protein  solution.  Protein  struc- 
tures hardened  under  strain  form  radiating  structures  resembling 
centrosomes  and  the  radiating  threads  seen  in  cells.  If  elder  pith 
is  saturated  with  protein  solutions  and  then  hardened,  sectioned, 
and  stained  by  the  usual  methods,  appearances  resembling  closely 
the  structure  of  a  hardened  cell  may  be  found  in  the  spaces  of  the 

«  Pentamalli,  Arch.  Entwick.  u.  Org.,  1912  (34),  444;  McClendon,  Proc.  Soc. 
Exp.  Biol,  and  Med.,  l'.)l()  (7),  111;  Hardy,  .lour.  Pliysiol.,  1913  (47),  lOS. 


CELL  STRUCTURE  43 

l)ith — even  a  (-(Mitral,  iiuclcus-likc  mass  may  be  suspencJcd  in  a  net- 
work of  anastomosing  threads.  These  and  many  other  experi- 
ments indicate  that  much  of  tlie  work  done  on  cell  structure  by  means 
of  studies  of  hardened  cells  cannot  be  considered  of  value  in  deciding 
the  structure  of  living  cells;  but,  nevertheless,  the  fact  remains  that 
man}^  cells  that  can  be  observed  while  alive  and  uninjured  under  the 
microscope  are  seen  to  have  a  definite  structure  in  the  cytoplasm, 
e.  g.,  sea-urchin  eggs,  which  show  a  characteristic  alveolar  structure. 

A  compromise  view  of  the  structure  of  protoplasm  (and  cytoplasm 
in  particular)  which  takes  account  of  what  appear  to  be  facts  brought 
out  on  both  sides  of  the  question,  is  that  while  in  some  cells  definite 
structural  arrangements  of  the  cytoplasm  exist,  in  most  cells  the 
proteins  are  chiefly  in  a  homogeneous  solution;  most  of  the  structures 
seen  in  fixed  cells,  except  the  mitochondria,  chromatin  threads,  nuclear 
membrane,  nucleoli,  and  centrosomes,  are  produced  by  the  coagulation 
of  the  proteins,  and  are  not  present  during  life.  When  a  framework 
does  exist,  it  is  a  fair  inference,  by  analogy  with  the  cell  mem- 
brane and  the  stroma  of  the  red  corpuscles,  that  the  cell  lipoids  are 
largely  responsible  for  its  formation,  and  that  they  form  a  prominent 
part  of  its  composition.  This  question  of  the  presence  or  absence  of 
structure  in  the  cytoplasm  is  of  more  importance  than  as  a  mere  mor- 
phological problem,  for  if  the  cytoplasm  is  subdivided  into  innumer- 
able little  chambers,  each  surrounded  by  a  membrane,  it  is  probable 
that  processes  of  diffusion  and  conditions  of  osmotic  pressure  will  be 
very  different  from  what  they  would  be  if  the  cytoplasm  were  a  simple 
homogeneous  colloid  solution,  like  a  lump  of  semisolid  gelatin  or  agar. 
In  such  colloidal  masses  diffusion  and  osmosis  go  on  almost  as  if  there 
were  no  colloids  in  the  solvent  at  all,  whereas  most  membrane  struc- 
tures that  are  found  in  living  tissues  seem  to  have  a  decidedly  semi- 
permeable character. 

From  what  we  know  at  the  present  time  of  intracellular  physics 
and  chemistry  there  is  no  necessity  for  assuming  that  semipermeable 
septa  exist  within  the  cell.  All  the  intracellular  processes  with  which 
we  are  familiar  could  go  on  without  such  structures.  It  is  not  neces- 
sary to  assume  a  compartment  structure  to  explain  the  possibility  of 
different  chemical  reactions  going  on  in  different  parts  of  the  cell  at 
the  same  time,  for  most  of  the  cell  reactions  seem  to  depend  on 
enzymes,  which  we  know  are  not  readily  diffusible  in  solutions  of  col- 
loids, and,  therefore,  might  remain  fixed  without  requiring  any  en- 
closing walls  or  retaining  framework.  Certainly,  many  cells  are  free 
from  structural  cytoplasm,  for  we  see  particles  of  solid  matter  moving 
about  within  them  quite  freely.  In  some  cells  the  nuclei  migrate 
about  in  the  cell,  as  also  do  digestive  and  excretory  vacuoles,  which 
motion  would  seem  to  be  rather  destructive  if  the  protoplasm  had  a 
structure  at  all  permanent. 

When  a  portion  of  the  cytoplasm  is  cut  free  from  the  body  of 


44  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

certain  cells  it  at  once  forms  a  round  drop,  just  as  any  insoluble 
fluid  would  do  in  another  of  different  surface  tension,  and  not  at  all 
as  if  it  were  bound  into  a  fixed  structure  by  a  framework.  Other 
cells,  however,  retain  their  form  under  the  same  conditions.  The 
structure  of  even  so  evidently  complicated  a  cytoplasm  as  that  of 
striated  muscle  fibers  is  in  doubt;  a  classical  observation  on  this  point 
is  the  passage  of  a  minute  worm  through  the  substance  of  a  muscle- 
cell,  its  progress  being  as  unimpeded  as  if  there  were  no  such  things 
as  disks,  bands,  rods,  and  striae  in  the  cell.  Many  features  of  ame- 
boid movement  also  seem  to  indicate  that  the  cytoplasm  follows 
much  the  same  laws  as  a  drop  of  fluid  in  a  heterogeneous  medium,  for 
we  can  make  a  drop  of  mercury  or  of  chloroform  in  water,  or  of  oil 
in  weak  alcohol,  react  to  various  stimuli  in  much  the  same  waj'  that 
an  ameba  would.  If  we  look  upon  the  cytoplasm  as  a  drop  of  emulsion 
colloid,  the  surfaces  of  the  particles  in  the  emulsion  furnish  of  them- 
selves adequate  explanation  of  many  of  the  phenomena  of  isolation 
of  chemical  reactions,  etc.,  without  lacking  in  harmony  with  the  evi- 
dences of  structural  homogeneity.  This  hypothesis  fits  all  sides  of 
the  problem  and  has  many  supporters  at  the  present  time.*^ 

The  question  of  structure  in  the  nucleus  is  quite  a  different  matter, 
in  so  far  as  the  chromatin  threads  and  the  nucleolus  are  concerned.  In 
ameboid  movement  the  nucleus  seems  to  play  a  passive  role  and  to 
be  dragged  about  by  the  cytoplasm,  indicating  quite  a  high  degree  of 
rigidity.  It  is  probable,  however,  that  the  achromatic  portion  between 
the  chromatin  threads  and  granules  has  much  the  same  structure  or 
lack  of  structure  as  the  cytoplasm. 

The  various  secretory  granules,  fat-droplets,  pigment-granules,  glycogen  gran- 
ules, keratin,  etc.,  that  may  lie  in  the  cytoplasm,  are  inconstant  constituents, 
varying  with  different  cells,  and  under  varying  conditions  in  the  same  cells,  and 
lie  beyond  the  scope  of  our  discussion  of  the  general  composition  of  the  cell.  Ac- 
cording to  Ruzicka'*^  there  is  contained  in  all  cells,  both  in  nucleus  and  cytoplasm, 
an  insoluble  substance  which  corresponds  structurally  to  the  "plastin"  of  the 
cytologists,  and  chemically  is  related  to  the  reticulins  and  other  albuminoids; 
this  he  looks  upon  as  the  ground  substance  of  the  cells,  corresponding  to  the  albu- 
minoid ground  substance  or  stroma  of  organized  tissues. 

Certain  of  the  granulations  observed  in  the  cj^toplasm  of  cells  seem  to  be  de- 
finite, constant  structures  of  the  living  protoplasm,  and  these  are  now  called  mito- 
chondria, which  term  includes  many  forms  of  granules  described  \mder  various 
names."  Their  solubility  and  staining  reactions  suggest  that  they  contain  phos- 
pholipins,  perhaps  associated  with  proteins.  Their  functional  importance  is  in- 
dicated by  the  fact  that  usually  their  number  varies  directly  with  the  metabolic 
activity  of  the  cells,  and  they  may  lie  related  to  histogenesis. 

Other  histological  cellular  structures  also  permit  of  more  or  less  satisfactory 
identification  by  microchemical  methods,  and  Unna""'  especially  has  contributed 
to  this  field.  By  staining  sections  with  dyes  of  varying  reaction,  after  extracting 
the  sections  with  various  solvents,  he  has  obtained  evidence  of  the  chemical  nature 

"An  excellent  discussion  of  this  question  is  given  bv  Alslierg,  Science,  1911 
(34),  97. 

**  Arch.  f.  Zellforsch.,  1908  (1),  5S7. 

«  Sec  review  bv  Cowdry,  Amer.  .lour.  Anat.,  191()  (19),  423;  Carnegie  Inst. 
Publ.,  No.  2."),  1918. 

"  See  review  by  Gans,  Dcut.  med.Woch.,  1913  (39),  1944. 


CELL  STRUCTURE  45 

of  some  of  the  cell  structures,  although  it  is  by  no  means  certain  that  the  conclu- 
sions drawn  will  all  be  verified.  In  the  nucleolus  he  finds  a  substance  resembling 
glol)uliu,  the  i^runuloplasiu  of  tlic  cell  body  lie  rej^ards  as  an  albumose,  the  spongio- 
plasni  as  histone,  mast  cell  granules  as  mucin  or  mucoid  substances.  Nissl  bodies 
he  holds  to  be  albumose,  altho  otiiers  have  beUeved  them  to  be  nucleins." 

The  Cell-wall** 

The  cell  membrane  in  most  animal  cells  is  inconspicuous  struc- 
turally, but  in  discussing  osmosis  it  was  shown  that  it  is  of  the  greatest 
biological  importance.  There  is  no  direct  chemical  or  microscopical 
evidence  at  hand  showing  the  composition  of  the  animal  cell  mem- 
brane, but  by  observations  on  its  behavior  when  the  cells  are  in  solu- 
tions of  different  sorts,  facts  have  been  collected  indicating  that 
phospholipins  and  cholesterol,  and  probabl}^  alhed  fat -like  bodies,  are 
prominent  constituents.  The  substances  that  difTuse  through  most 
cell  walls  are  just  the  substances  that  are  soluble  in  or  dissolve  these 
lipoids,  e.  g.,  alcohol,  chloroform,  ether,  etc.,  and  it  is  probable  that 
the  anesthetic  effects  of  many  of  these  substances  depend  in  some  way 
on  their  fat-dissolving  power  and  the  large  proportion  of  lipoids  in 
nerve-cells.  These  observations  were  made  first  by  Overton^^  and 
Meyer,  ^"^  and  led  to  the  now  prominent  but  disputed  hypothesis  that  the 
permeability  of  cells  is  determined  by  the  lipoids.  Of  particular  in- 
terest for  our  purpose  are  Overton's  observations  on  the  effects  of  dyes 
on  living  cells.  The  best  known  vital  stains  (z.  e.,  stains  that  will 
enter  the  living  cell  without  requiring  or  causing  injury  to  it)  are  neu- 
tral red,  methylene  blue,  toluidin  blue,  thionin,  and  safranin.  If 
uninjured  cells,  e.  g.,  frog  eggs,  are  placed  in  watery  solutions  of  these 
dyes  they  soon  become  filled  with  the  coloring-matter,  which  seems  to 
penetrate  the  cell  menbrane  quite  uniformly  at  all  points;  if  the  dyed 
eggs  are  then  placed  in  clear  water,  the  stain  diffuses  out  again,  showing 
it  to  be  simply  absorbed,  rather  than  chemicallj^  combined.  In 
contrast  to  these  stains  the  sulphonic  acid  dyes,  such  as  indigo  car- 
mine and  water-soluble  indulin,  nigrosin,  and  anilin  blue,  do  not  pene- 
trate the  living  cell  at  all.  Overton  tested  the  solubilit  j^  of  dyes  which 
are  not  vital  stains  and  found  them  all  insoluble  in  oils,  fats,  and  fatty 
acids;  but  the  dyes  staining  living  cells  were  readily  soluble  in  lecithin, 
cholesterol,  "protagon,"  and  cerebrin,  the  so-called  cell  lipoids.  Fur- 
thermore, if  crumbs  of  lecithin,  "protagon,"  or  cerebrin  were  placed 
in  very  dilute  watery  solutions  of  these  dyes,  they  were  found  to  absorb 
from  the  water  the  vital  stains,  but  not  the  others,  which  indicates 
that  stains  that  penetrate  living  cells  are  more  soluble  in  lipoids  than 
they  are  in  water. 

"  See  Unna,  Berl.  klin.  Woch.,  1914  (51),  444;  Muhlmann,  Arch.  mikr.  Anat., 
1914  (85^  ,361. 

•**  See  Zangger,  "Ueber  Membranen  und  Membranenfunktionen,"  Ergebnisse  d. 
Physiol.,  1908  (7),  99;  also  R.  S.  Lillie,  "The  Role  of  Membranes  in  Cell  Pro- 
cesses," Popular  ScienceMonthlv,  Feb.,  1913. 

"  Jahrb.  f.  wissentschaftl.  Botanik,  1900  (34),  669. 

"  Arch.  f.  exp.  Path.  u.  Pharm.,  1899  (42),  109. 


46  THE  CHEMISTRY  AND  PHYSICS  OF  THE  CELL 

Many  exceptions  to  this  rule  of  the  fat-solubihty  of  dyes  which 
can  penetrate  Hving  cells  have  been  found,  especially  by  Ruhland,^^ 
and  the  universal  applicabihty  of  the  Overton-Meyer  hypothesis  has 
been  questioned.  It  is  at  once  evident  that  the  common  foodstuffs 
which  enter  the  cell,  such  as  water,  sugar,  amino-acids,  and  salts  are 
not  lipoid-soluble,  hence  it  has  been  suggested  that  the  cell  membranes 
must  have  a  "mosaic"  structure,  some  of  the  blocks  being  lipoids  or 
lipoid  compounds,  and  others  proteins  without  lipoids.  (Robertson*^ 
suggests  that  there  is  a  superficial  film  of  concentrated  protein  about 
the  cells,  underlaid  by  a  discontinuous  lipoid  layer.)  There  is,  fur- 
thermore, evidence  that  the  entire  cell  substance  has  a  profound  effect 
upon  diffusion  within  the  cell,  so  that  it  is  at  present  impossible  to 
say  whether  the  osmotic  phenomena  of  cells  depend  upon  a  cell  mem- 
brane or  upon  the  entire  cell  substance. ^^  It  may  be  that  there  are 
membranes  or  surfaces  within  the  cell,  as  postulated  in  the  foam 
structure  hypothesis  of  protoplasm,  or  that  a  homogeneous  protoplasm 
develops  surfaces  where  in  contact  with  substances  entering  from  the 
outside. 

Many  facts  indicate  that  either  the  delicate  external  membrane 
of  animal  cells  or  the  entire  cytoplasm  has  the  features  of  a  semi- 
permeable membrane,  to  the  extent  of  permitting  certain  substances 
to  diffuse  through  and  not  others.  Had  they  the  property  of  some 
of  the  artificial  semipermeable  membranes,  of  letting  water  pass 
through  but  holding  back  almost  absolutely  all  crystalloids,  the  re- 
sult would  be  the  development  of  an  enormous  disproportion  in  the 
pressure  between  the  inside  and  the  outside  of  the  cell.  Furthermore, 
the  exchange  of  nutritive  material  and  excretion  products  between  the 
blood  and  the  cells  would  be  impossible.  But  permitting  some  sub- 
stances to  pass  into  the  cell  results  in  their  accumulation  within  the 
cell,  until  they  are  in  sufficient  concentration  to  neutralize  the  osmotic 
pressure  exerted  on  the  outside  of  the  cell.  As  evidence  of  this  elec- 
tive permeability  we  have  the  fact  that  the  proportion  of  certain  salts 
within  the  cell  is  quite  different  from  what  it  is  in  the  fluids  bathing 
them;  e.  g.,  animal  cells  generally  contain  more  potassium  and  less 
sodium  than  the  fluids  surrounding  them.  The  inorganic  constituents 
of  red  cells  are  different  from  those  of  the  plasma,  the  corpuscles 
not  containing  any  calcium  at  all,  while  the  magnesium  seems  to 
enter  them  freely;  in  other  words,  the  red  corpuscle  seems  to  be 
impermeable  to  calcium  and  permeable  to  magnesium.  If  the  salts 
in  a  corpuscle  are  in  smaller  proportions  than  in  the  surrounding  fluid, 
it  indicates  that  the  cell  membrane  is  not  freely  permeable  for  them; 
if  in  greater  proportion,  that  some  constituent  of  the  cell  is  holding 
them  in  combination,  possibly  as  ion-protein  compounds.     Probably 

6'  Jahrb.  f.  Wisscnschaft.  Botanik,  1912  (51),  376. 

'•■'Jour.  Biol.  CluMn.,  1908  (4),  1. 

"  Sec  Kite,  Aincr.  Jour.  Thysiol.,  1915  (37),  282;  Chambers,  ibid.,  1917  (-13),  1. 


CELL  STRUCTURE  47 

inorganic  salts  are  present  in  the  cell  by  virtue  of  both  physical  and 
chemical  influences,  some  simply  (Hffusiii^;  in  and  out,  others  com- 
bining with  the  proteins  and  being  held  chemically. 
Bechhold  summarizes  his  conception  of  cell  walls  as  follows: 
"Every  cell  at  its  surface  possesses  a  membrane  which  is  dependent 
upon  the  composition  of  the  interior  of  the  cell.  This  membrane 
may  be  visible  and  may  have  been  formed  through  the  gelatinizal  ion  of 
the  cell  protoplasm  at  the  periphery.  It  may,  on  the  other  hand,  be  so 
thin  as  to  be  invisible,  being  formed  by  the  concentration  and  spread- 
ing out  of  such  albuminous  and  fatty  colloids  as  diminish  the  surface 
tension  of  the  cell  content  at  the  interface.  The  cell  membranes,  de- 
veloping as  a  result  of  the  gelatinization  of  cell  protoplasm,  are  at 
first,  in  youth,  expansile  and  elastic;  with  increasing  age  these  mem- 
brane colloids,  depending  upon  their  environment  and  upon  chemical 
influences,  or  as  a  result  of  mere  colloid  aging  phenomena,  become  poor 
in  water  and  lose  their  elasticity." 

The  intercellular  substance  varies  greatlj^  in  different  tissues.  In  the  case  of 
the  supportive  tissues  it  is  the  important  element,  and  the  cells  seem  to  exist  chiefly 
for  the  purpose  of  forming  and  keeping  it  in  repair  as  it  is  worn  out.  In  the  epi- 
thelial and  secreting  tissues,  however,  the  intercellular  substance  is  reduced  to  a 
minimum,  except  in  so  far  as  a  cement  substance  is  required,  and  the  cells  generally 
lie  in  almost  immediate  apposition.  It  is  probable  that  there  is  a  greater  or  less 
amount  of  cement  substance,  even  between  the  most  closely  applied  cells,  and  this 
substance  seems  to  be  related  to  mucin.  It  can  generally  be  brought  out  by  stain- 
ing with  silver  nitrate,  and  Macallum^^  points  out  that  this  reaction  is  merely  a 
micro-chemical  test  for  chlorides,  and  indicates  that  the  cement  substance  con- 
tains them  in  larger  proportion  than  does  the  cytoplasm. 

"  Proceedings  of  the  Royal  Society,  1905  (76),  217. 


CHAPTER  II 

ENZYMES 

Every  cell  is  constantly  accomplishing  an  enormous  number  of 
chemical  reactions  of  varied  natures,  at  one  and  the  same  time;  how 
many  we  do  not  know,  but  the  score  or  more  that  we  do  know  to  be 
constantly  going  on  in  the  liver  cell,  for  example,  are  probably  but 
a  part  of  the  whole.  Furthermore,  reactions  take  place  between  sub- 
stances that  show  no  inclination  to  affect  each  other  outside  the  body, 
and  proceed  in  directions  that  we  find  it  difficult  to  make  them  take 
in  the  laboratory.  Proteins  are  being  continually  broken  down  into 
urea,  carbon  dioxide,  and  water;  yet  to  split  proteins  even  as  far  as  the 
amino-acid  stage  requires  prolonged  action  of  concentrated  acids  or 
alkalies,  or  super-heated  steam  under  great  pressure.  But  all  the  time 
in  the  cell  innumerable  equally  difficult  changes  are  going  on  at  once, 
within  its  tiny  mass,  always  keeping  the  resulting  heat  within  a  frac- 
tion of  a  degree  of  constant,  and  the  resulting  products  within  narrow 
limits  of  concentration.  We  have  already  indicated  the  means  used 
to  keep  the  concentration  of  the  cell  products  within  safe  limits; 
namely,  the  processes  of  diffusion  and  osmosis  and  their  modification 
by  the  cell  structure.  The  forces  that  bring  about  the  chemical  reac- 
tions reside,  we  say,  in  enzymes,  although  in  so  doing  we  only  shift 
the  attribute  formerly  conceded  to  the  cell,  to  certain  constituents  of 
the  cell  whose  nature  and  manner  of  action  are  equallj^  unknown. 
When  the  only  enzymes  that  were  known  were  limited  to  those  se- 
creted from  the  cell,  and  found  free  in  fluids,  such  as  pepsin  and  tryp- 
sin, the  chemical  changes  that  went  on  in  the  cell  were  ascribed  to  its 
"vital  activity."  Buchner,  by  devising  a  method  to  crush  yeast  cells, 
and  finding  the  expressed  cell  contents  able  to  produce  the  same 
changes  in  carbohydrates  that  the  cells  themselves  did,  proved  the  ex- 
istence within  living  cells  of  enzymes  similar  to  those  excreted  by  cer- 
tain cells,  and  substantiated  the  belief  of  their  existence  that  had 
become  general  before  it  was  thus  finally  corroborated.  Growing  out 
from  this  and  subsequent  experiments  has  come  a  larger  and  larger 
amount  of  evidence  that  many  of  the  chemical  activities  of  the  cells 
are  due  to  the  enzymes  they  contain,  until  now  the  point  is  reached 
where  one  may  rightfully  ask  if  cell  life  is  not  entirely  a  matter  of 
enzyme  activity.  There  are  certain  facts,  however,  which  seem  to  in- 
dicate  that  there   are  some  essential  differences  between  cells  and 

48 


NATURE  OF  ENZYMES  40 

enzymes.  One  of  the  most  important  of  these  is  the  difference  in  the 
susceptibility  to  poisons  of  enzymes  and  cells. ^  Strengths  of  certain 
antiseptics  that  will  either  destroy  or  inhibit  the  action  of  living  cells, 
such  as  alcohol,  ether,  salicyhc  acid,  thymol,  chloroform  and  toluene, 
will  harm  free  enzymes  in  solution  little  or  not  at  all.  This  fact  has 
been  of  great  assistance  in  distinguishing  between  the  action  of  en- 
zymes and  of  possible  contaminating  bacteria  in  experimental  work. 
Although  this  difference  between  enzymes  and  cells  is  characteristic, 
it  does  not  finally  decide  that  the  cell  actions  are  not  enzyme  actions, 
for  it  may  well  be  that  the  poisons  act  chiefly  by  altering  the  physical 
conditions  of  the  cell  so  that  diffusion  is  interfered  with,  thus  seriously 
interfering  with  the  exchange  of  cleavage  products  between  different 
parts  of  the  cell,  and  checking  intracellular  enzyme  action,  which  we 
shall  see  later  requires  free  diffusion  of  the  products  for  its  continuance. 
At  the  very  least,  however,  we  may  look  upon  the  intracellular  en- 
zymes as  the  most  important  known  agents  of  cell  metabolism,  and 
consequently  of  all  life  manifestations,  and  the  changes  they  undergo 
or  produce  in  pathological  conditions  must  be  fully  as  fundamentally 
important  as  is  their  relation  to  physiological  processes.  It  therefore 
becomes  necessary  for  us  to  consider  carefully — 

'THE  NATURE  OF  ENZYMES  AND  THEIR  ACTIONS 

Since  up  to  the  present  time  no  ferment  has  been  isolated  in  an  absolutely  pure 
condition  we  are  entirely  unfamiliar  with  their  chemical  characters,  and  conse- 
quently are  obUged  to  recognize  them  solely  by  their  action.  As  far  as  we  know, 
true  enzymes  never  occur  except  as  the  result  of  cell  life — they  are  produced  with- 
in the  cell,  and  increased  in  amount  b}^  each  new  cell  that  is  formed,  and,  further- 
more, they  are  present  in  every  living  cell  without  exception.  As  the  same  facts 
are  equally  true  of  the  proteins  it  is  natural  to  associate  the  enzymes  with  pro- 
teins, and  so  explain  the  importance  of  the  proteins  for  ceU  life.^  If  enzymes  are 
obtained  in  any  of  the  usual  ways  from  animal  cells  or  secretions  they  are  always 
found  to  give  the  reactions  for  proteins,  even  if  repurified  many  times.  But  it 
is  well  known  that  whenever  proteins  are  precipitated  the  other  substances  in 
the  solution  tend  to  be  dragged  down  by  the  colloids,  and  it  is  possible  that  the  en- 
zymes are  merely  associated  with  the  proteins  in  this  way.  Furthermore,  enzymes 
are  known  to  become  so  closely  attached  to  stringy  protein  masses,  such  as  fibrin 
and  silk,  that  they  cannot  be  removed  by  washing.  Some  have  claimed  that  they 
have  secured  active  preparations  of  pepsin  and  invertase  that  did  not  give  protein 
reactions  and  contained  very  little  or  no  ash  or  carbohydrate ;  but  it  has  so  far  been 
impossible  to  secure  trypsin  free  from  protein,  and  diastase  seems  to  be  certainly  of 
protein  nature.     Davis  and  Merker^  find  that  the  more  pepsin  is  purified  the  more 

1  See  discussion  by  Vernon,  Ergebnisse  d.  Physiol..  1910  (9),  234. 

-  It  would  not  be  profitable  to  discuss  fully  all  the  various  theories  and 
hypotheses  that  have  been  advanced,  but  the  reader  is  referred  to  the  following 
chief  compilations  of  the  entire  subject:  Oppenheimer,  "Die  Fermente  und  ihre 
Wirkungen,"  Leipzig;  BayHss,  "The  Nature  of  Enzyme  Action,"  Monographs  on 
Biochemistry,  London;  Stern,  "Physico-chemical  Basis  of  Ferment  Action,"  in 
Oppenheimer's  "Handbuch  d.  Biochemie,"  Vol.  4,  pt.  2;  Samueley,  "Animal  Fer- 
ments," ibid,  Vol.  I;  A.  E.  Taylor,  "On  Fermentation,"  Univ.  of  California  I\ibUca- 
tions;  Euler,  "General  Chemistry  of  the  Enzymes,"  translated  by  T.  H.  Pope, 
New  York,  1912. 

'  Another  important  point  is  that  the  closest  imitation  of  enzymes,  Bredig's 
'inorganic  ferments,"  seem  to  owe  their  action  to  their  colloidal  nature. 

*  Jour.  Amer.  Chem.  Soc,  1919  (41),  221. 

4 


50  ENZYMES 

it  approaches  the  character  of  a  protein,  possibly  a  glycoprotein,  with  increasing 
proteolytic  activity.^  Analyses  of  enzymes  purified  as  completely  as  possible 
do  not  have  great  worth,  for  the  "purified"  enzymes  are  probably  far  from  pure; 
however,  it  is  of  some  importance  that  the.y  vary  greatly  in  the  proportions  of 
carbon,  hydrogen,  and  nitrogen  which  they  contain,  indicating  that  possibly  dif- 
ferent enzymes  may  be  of  very  different  nature.  The  enzjanes  have  been  found 
to  possess  definite  electrical  charges;  in  neutr-il  solutions  trypsin  is  negative  or 
amphoteric,  pepsin  and  invertase  negative  (Michaelis).^  Macallum  has  shown 
microchemically  that  phosphorus  is  closely  associated  with  the  formation  of 
zymogen  granules  in  cells,  which  seem  to  be  started  in  the  nucleus;  and  there  are 
many  other  observations  suggesting  that  certain  ferments  are  closely  related  to 
the  nucleo-proteins.  This  is  particularly  true  of  the  oxidases,  which  seem  also 
to  contain  iron  and  inanganese.  A  final  point  of  importance  in  support  of  the 
protein  nature  of  enzymes  is  that  pepsin  destroj^s  trypsin  and  diastase,  while 
trypsin  destroys  pepsin.' 

So  uncertain,  however,  is  our  information  concerning  the  chemical  nature  of 
the  enzymes,  that  it  has  become  possible  for  an  hypothesis  to  be  developed  urg- 
ing that  enzymes  are  immaterial,  that  the  actions  we  consider  as  characterizing 
enzymes  are  the  result  of  physical  forces  which  may  reside  in  many  substances, 
and  perhaps  even  free  from  visible  matter,  but  the  weight  of  evidence  at  present 
available  is  entirely  in  favor  of  the  view  that  enzymes  are  specific  colloidal  sub- 
stances, although  perhaps  of  widely  differing  chemical  nature.  A  valuable  piece 
of  evidence  of  the  material  existence  of  enzymes  is  their  specific  nature,  lipase 
affecting  only  fats,  and  trypsin  only  proteins,  indicating  chemical  individuality. 
They  are  true  secretions,  formed  within  the  cell  by  recognizable  steps;  and, 
furthermore,  when  injected  into  the  body  of  an  animal,  they  give  rise  to  the  forma- 
tion of  specific  immune  bodies  that  antagonize  their  action.  Emil  Fischer's 
work  with  the  sugar-splitting  enzymes,  moreover,  indicates  that  they  owe  their 
action  to  their  stereochemical  configuration.  He  prepared  two  sets  of  sugar  de- 
rivatives which  differed  from  each  other  solely  in  the  arrangement  of  their  atoms 
in  space  (i.  e.,  isomers)  and  found  that  one  specific  enzyme  would  split  members  of 
only  one  of  the  varieties,  while  another  enzyme  would  act  only  on  the  variety 
with  the  opposite  isomeric  form.  These  experiments  make  it  very  probable  that 
there  must  be  a  certain  relation  of  geometrical  structiu-e  between  an  enzyme  and 
the  substances  it  acts  upon,  and  leaves  little  question  of  its  material  nature. 

Bredig  has  found  that  colloidal  solutions  of  metals  have  many  of  the  properties 
of  true  enzymes,  accomplishing  many  of  the  decompositions  produced  by  en- 
zymes, being  affected  by  temperatures  of  nearly  the  same  degree,  and  even  being 
"poisoned"  by  substances  that  destroy  or  check  enzj^mes.^  The  only  possible 
explanation  of  these  observations  seems  to  be  that  the  enzyme  effects  are  brought 
about  by  surface  -phenomena.  A  colloidal  solution  of  platinum,  as  far  as  is  known, 
differs  from  a  piece  of  metallic  platinum  solely  in  the  enormously  great  amount  of 
surface  it  offers  in  proportion  to  its  weight,  and  it  is  well  known  that  surfaces  may 
affect  chemical  action.  Hence  we  have  the  possibility  that  some  enzyme  actions, 
at  least,  may  depend  upon  the  existence  of  a  very  large  surface,  and  since  by  no 
means  all  colloids  are  enzymes,  that  this  surface  must  bear  a  certain  relation  in 
form  to  the  surface  of  the  body  that  is  to  be  acted  upon. 

The  Principles  of  Enzyme  Action 

The  effects  produced  by  enzymes,  which  at  one  time  were  con- 
sidered quite  unique  and  remarkable,  have  no\v  been  made  compara- 
tively plain,  chiefly  through  the  observations  of  Ostwald  on  related 

5  Bokorny  (Biochem.  Zeit.,  1919  (94),  G9)  finds -that  the  amount  of  formalde- 
hyde fixed  by  emulsin  supports  the  hypothesis  that  this  enzyme  is  a  protein. 

"  Bioclieni.  Zeit.,  1909  (l(j),  81  and  480;  (17),  231. 

'  Falk  lias  ol)tained  evidence  that  ester-splitting  enzymes  may  be  proteins 
owing  their  activity  to  the  presence  in  the  molecule  of  active  groupings,  perhaps 
of  enol-lactim  structure,  — C(OII)  =  N — .  B.y  treating  pure  proteins  with  alkali, 
which  favors  the  fornuition  of  enol-lactim  groupings,  the  proteins  were  made  to 
acquire  esterase  properties.      (See  Science,  1918  (47),  423.) 

"  See  also  Fischer  and  Hooker,  J.  Lab.  Clin.  Mini.,  1918  (3),  373. 


ENZYME  ACTION  51 

c;ieniical  reactions;  ami  by  the  investigations  of  Croft  Hill,  Kastle 
and  Loevenhart,  and  others,  on  enzymes,  which  show  that  enzyme 
action  is  in  no  way  different  from  chemical  action  observed  independ- 
ent of  enzymes.  The  fundamental  consideration  is  that  chemical  re- 
actions are  reversible,  that  is,  that  their  tendency  is  to  establish  an 
equilibrium,  and  that  the  change  may  be  from  either  side  of  the  equa- 
tion.^ The  action  of  enzymes  is  similar  to  that  of  all  catalytic  agents, 
that  is,  they  increase  the  speed  of  reaction.  In  the  case  of  such  a 
reaction  as  that  of  NaOH  and  HCl,  the  reaction  is  so  rapid  that  the 
effect  of  catalyzers  could  hardly  be  noticed;  but  with  many  other 
substances  the  reaction  is  very  slow,  and  without  the  presence  of 
catalyzers  it  would  go  on  almost  or  quite  imperceptibly.  For  ex- 
ample, ethyl  butj'rate  saponifies  on  the  addition  of  water  according 
to  the  following  equation: 

C.H5  -  O  -  OC  -  C3H7  +  H2O  ^  C0H5OH  +  HOOC  -  C3H7. 

On  the  other  hand,  if  ethjd  alcohol  and  butj-ric  acid,  the  products 
of  this  reaction,  are  placed  together,  they  will  combine  to  form  ethyl 
butyrate;  in  other  words,  the  reaction  is  reversible,  as  indicated  by 
the  arrows  in  the  equation.  In  any  event,  however,  the  reaction  is  not 
complete,  but  continues  only  until  there  exists  a  certain  definite  pro- 
portion of  ethyl  alcohol,  butyric  acid,  eth3'l  butyrate,  and  water,  when 
the  change  will  stop,  i.  e.,  equilibrium  is  established.  The  time  that 
would  be  required  for  this  reaction  to  occur  at  room  temperature 
would  be  extremely  long,  the  change  being  hardly  noticeable,  but  in 
the  presence  of  a  catalytic  agent  the  reaction  goes  on  much  more 
rapidly.  Catalytic  agents,  therefore,  merely  hasten  reactions  which 
would  go  on  without  them,  and  they  do  not  initiate  or  change  the  na- 
ture of  chemical  reactions  at  all.  When  equilibrium  is  established,  the 
reaction  stops  and  the  enzyme  has  nothing  more  to  do.  Furthermore, 
enzj^mes  will  hasten  synthesis  just  as  well  as  they  hasten  catalysis. 
Croft  Hill  first  showed  that  maltase  would  synthesize  glucose  intc 
maltose;  Kastle  and  Loevenhart  soon  after  estabhshed  the  synthesis 
of  ethyl  butyrate  under  the  influence  of  lipase.  Taylor^ °  first  syn- 
thesized one  of  the  normal  body  fats,  triolein,  by  the  action  of  hpase 
(from  the  castor-oil  bean)  upon  oleic  acid  and  glycerol.  Successful 
synthesis  of  fats  by  pancreatic  lipase  is  described  by  Lombroso.^^ 
It  may  seem  improbable  at  first  sight  that  the  synthesis  of  proteins 
can  be  accomplished  by  enzymes,  as  is  the  relatively  very  simple 
synthesis  of  carbohydrates  and  fats,  but  the  improbability  disappears 
when  we  recall  that  the  products  of  protein  cleavage  are  reconverted 
into  body  proteins  after  absorption  from  the  intestines.  Proteins 
manifestly  are  synthesized  and  we  have  not  a  little  reason  to  believe 

9  See  Taylor,  Arch.  Int.  Med.,  190S  (2),  148. 
i»  Univ.  of  California  Publications  (Pathology),  1904  (1),  33. 
11  .\rch.  di  farmacol.,  1912  (14),  429. 


52  ENZYMES 

that  this  is  accompUshed  by  enzymes,  presumably  by  a  reversal  of 
their  action  in  the  establishment  of  equilibrium.  Abderhalden^^  has 
obtained  some  evidence  of  protein  formation  in  mixtures  of  amino 
acids  derived  from  autolyzing  tissue  when  acted  upon  by  ferment- 
containing  extracts  of  the  same  tissue.  Taylor^^  was  able  to  synthesize 
protamin,  one  of  the  simplest  proteins,  by  the  action  of  trypsin  upon 
its  cleavage  products,  and  it  has  been  found  that  the  addition  of 
proteolytic  enzymes  to  solutions  of  pure  albumose  leads  to  the  forma- 
tion of  a  jelly-like,  insoluble  protein  substance,  "  plastein,"  which  seems 
to  be  the  effect  of  a  reversed  action  on  the  part  of  the  enzymes. ^^ 
Another  well  known  synthetic  action  that  seems  to  be  due  to  reversible 
ferment  action  is  the  formation  of  hippuric  acid  from  benzoic  acid  and 
glycine  in  the  Iddney;  the  formation  of  glucose  into  glycogen  and  its 
reformation  are  also  probably  both  accomplished  by  one  and  the  same 
enzyme  acting  reversibly.  Other  reversible  reactions  less  closely 
related  to  animal  cells  have  also  been  described. 

The  reversible  nature  of  enzyme  action  explains  many  problems  of 
metabolism,  and  makes  the  whole  field  much  clearer.  The  following 
consideration  of  the  newer  understanding  of  fat  metabolism  on  this 
basis  may  explain  the  manner  in  which  chemical  changes  are  believed 
to  occur  in  the  cells  and  fluids  of  the  body:^^ 

In  the  intestines  fat  is  split  by  lipase  into  a  mixture  of  fat,  fatty  acid,  and 
glycerol;  but  as  the  fatty  acid  and  glycerol  are  diffusible,  while  the  fat  is  not, 
they  are  separated  from  the  fat  by  absorption  into  the  wall  of  the  intestine. 
Hence  an  equilibrium  is  not  reached  in  the  intestine,  so  the  splitting  continues 
until  practically  all  the  fat  has  been  decomposed  and  the  products  absorbed. 
When  this  mixture  of  fatty  acid  and  glycerol  first  enters  the  epithelial  cells  Uning 
the  intestines  there  is  no  equilibrium,  for  there  is  no  fat  absorbed  with  them  as 
such.  Therefore  the  lipase,  which  Kastle  and  Loevenhart  showed  was  present 
in  these  cells,  sets  about  to  establish  equilibrium  by  combining  them.  As  a  result 
we  have  in  the  cell  a  mixture  of  fat,  fatty  acid,  and  glycerol,  which  will  attain 
equilibrium  only  when  new  additions  of  the  two  last  substances  cease  to  enter 
the  cell.  Now  another  factor  also  appears,  for  on  the  other  side  of  the  cell  is 
the  tissue  fluid,  containing  relatively  little  fatty  acid  and  glycerol.  Into  this  the 
diffusible  contents  of  the  cell  will  tend  to  pass  to  establish  an  osmotic  equilibrium, 
which  is  quite  independent  of  the  chemical  equilibrium.  Tliis  abstraction  of  part 
of  the  cell  contents  tends  to  again  overthrow  chemical  equilibrium,  there  now  being 
an  excess  of  fat  in  the  cell.  Of  course,  the  lipase  will,  under  this  condition,  ex- 
hibit the  reverse  action  and  split  the  fat  it  has  just  built  into  fatty  acid  and  glycerol. 
It  is  evident  that  these  processes  are  all  going  on  together,  and  that,  as  the  composi- 
tion of  the  contents  of  the  intestines  and  of  the  blood-vessels  varies,  the  direction 
of  the  enzyme  action  mil  also  vary.  In  the  blood-serum,  and  also  in  the  lym- 
phatic fluid,  there  is  also  lipasC;  which  will  unite  part  of  the  fatty  acid  and  glycerol, 

'2  Fenuoritforschung,  1914  (1),  47. 

"  Jour.  Biol.  Chem.,  1909  (5),  381. 

1^  See  Michcli,  Arch.  ital.  biol,  190G  (46),  185;Levene  and  Van  Slvke,  Biochem. 
Zeit,  1908  (13),  458;  Taylor,  Jour.  Biol  Chem.,  1909  (5),  399;  Gav  and  Robert- 
son, ibid.  1912  (12),  233;  Alxlerhalden,  IVrnientforsch.,  1914  (1),  47;  v.  Knaffl- 
Lenz  and  Pick,  Arcli.  exp.  Path.,  1913  (71),  29(),  407. 

'5  See  Loevenhart,  Amer.  Jour,  of  Fliysiol.,  1902  (0),  331;  Wells,  Journal 
Amer.  Med.  Assoc,  1902  (38),  220.  The  discrepancies  between  the  action  of 
lipase  in  the  tissues  and  in  vitro  are  well  explained  by  Taylor,  Jour.  Biol.  Chem., 
1906  (2),  103. 


ENZYME  ACTION  53 

and  by  rcinovinf:;  tlicm  from  the  fluid  about  the  cells  favor  osmotic  diffusion  from 
tlie  intestinal  epithelium,  thus  facilitating  absorption. 

Quite  similar  must  be  the  process  that  takes  place  in  the  tissue  cells  through- 
out the  body.  In  the  blood-serum  bathing  the  cells  is  a  mixture  of  fat  and.  its 
constituents,  probably  nearly  in  equilibrium,  since  lipase  accompanies  them.  If 
the  diffusible  substances  enter  a  cell  containing  lipase,  c.  r/.,  a  liver  cell,  the  process 
of  building  and  splitting  will  be  quite  the  same  as  in  the  intestinal  epithelium. 
The  only  difference  is  that  here  the  fatty  acid  maj'  be  removed  from  the  cell  by 
being  utilized  by  oxidation  or  some  other  chemical  transformation.'* 

To  summarize,  it  may  be  stated  that  thiouglK)iit  the  body  there  is 
constantly  taking  place  both  splitting  and  building  of  fat.  Fat  enters 
the  cells,  leaves  them,  and  is  utilized  only  in  the  form  of  its  acid  and 
alcohol,  never  as  the  fat  itself.  Fat  constitutes  a  resting  stage  in  its 
own  metabolism. 

If  proteolytic  enzymes  also  act  reversibly,  then  the  phenomena  of 
protein  metabolism  are  similarly  explained,  for  there  is  no  doubt 
that  every  cell  and  body  fluid  contains  proteolytic  enzymes. 

All  metabolism,  then,  may  be  considered  as  a  continuous  attempt  at 
establishment  of  equilibrium  by  enzymes,  perpetuated  by  prevention  of 
attainment  of  actual  equilibrium  through  destruction  of  some  of  the 
participating  substances  by  oxidation  or  other  chemical  processes,  or  by 
removal  from  the  cell  or  entrance  into  it  of  materials  which  overbalance 
one  side  of  the  equation. 

In  just  what  manner  the  enzymes  accomplish  their  catalytic  effect 
is  yet  unknown.  1^  A  favorite  idea  is  that  they  form  loose  compounds 
with  the  substance  to  be  split  and  with  water;  the  resulting  compound 
being  unstable  and  breaking  down,  the  water  remains  attached  to 
the  components  of  the  substance. 

Enzymes  do  not  act  catalytically  on  all  substances  by  any  means, 
but  show  a  decidedly  specific  nature.  They  affect  only  organic  sub- 
stances, and  the  actions  are  limited  to  two  processes — hydrolj'sis  and 
oxidation,  or  the  reverse  processes  of  dehydration  and  reduction.'^ 
The  most  essential  difference  between  the  enzymes  and  the  chemicals 
that  can  accomplish  hydrolysis  or  oxidation  is  this:  the  ordinary 
chemical  reagents  produce  their  effects  on  many  sorts  of  substances, 
whereas  the  enzymes  are  specific;  thus  hydrochloric  acid  will  hydrolyze 
starch  or  protein  with  equal  facility,  but  pepsin  will  not  affect  starch 
at  all. 

The  very  specific  nature  of  the  enzymes,  their  activation  by  other 
body  products,  the  fact  that  they  seem  to  be  bound  to  the  substance 

16  Bradley  (Jour  Biol.  Chem.,  1910  (S),  251;  1913  (13),  407-439)  calls  atten- 
tion to  the  great  concentration  necessary  for  fat  synthesis  by  lipase  in  vitro,  and 
the  lack  of  correspondence  between  the  amount  of  fat  and  of  lipase  in  various 
tissues,  questioning  the  importance  of  lipase  for  fat  synthesis  in  the  living  tissues 
as  well  as  the  significance  of  reversed  enzyme  reaction  for  biological  processes  in 
general. 

'^  See  Euler,  "Chemical  Dynamics  of  Enzyme  Reactions,"  Ergebnisse  d.  Physiol. 
1910  (9),  241. 

18  Alcoholic  fermentation  may  be  an  exception,  the  change  being  CeHnOs  = 
2C2H6OH  -|-  2CO2,  but  it  is  very  possibly  an  intramolecular  oxidation. 


54  ENZYMES 

upon  which  they  act,  that  they  are  susceptible  to  heat,  and  that  they 
produce  immune  bodies  when  injected  into  experimental  animals,  all 
suggest  the  probability  of  a  relationshij)  between  enzymes  and  toxins. 
This  matter  will  be  discussed  more  fully  in  considering  the  chemistry 
of  immunity  against  enzjanes. 

General  Properties  of  Enzymes. — Other  properties  of  enzymes  may  be  briefly 
mentioned.  The  speed  of  reaction  they  produce  increases  with  the  amount  of 
enzymes  present.  Very  dihite  acids  favor  the  action  of  nearly  all  ferments,  and 
alkalies  are  unfavorable  for  all  but  trypsin,  ptyalin,  and  a  few  others.  Weak  salt 
solutions  also  are  more  favorable  than  distilled  water.  (These  facts  suggest 
strongly  the  possibility  that  ions  play  an  important  role  in  the  process.)  Water 
and  dilute  glycerol  dissolve  enzymes,  which  form  always  colloidal  solutions  that 
are  very  slightly  dialyzable;  and  they  may  be  precipitated  from  solution  by 
alcohol,  and  redissolved  again  with  but  sUght  impairment  of  strength.  Filtra- 
tion through  porcelain  filters  is  not  complete,  from  10  to  25  per  cent,  of  most  en- 
zymes being  lost  in  each  filtration  and  enzymes  are  subject  to  great  absorption  by 
surfaces,  e.  g.,  charcoal,  kaolin.^  As  before  mentioned,  many  chemicals  poison- 
ous to  bacteria  have  little  influence  on  most  enzymes,  but  nearly  all  substances 
when  concentrated  are  injurious  or  destructive,  and  some  enzymes  are  kno^^Ti 
that  are  more  susceptible  to  antiseptics  than  are  the  cells  that  contain  them. 
Formaldehyde  is  very  destructive  to  most  enzj^mes,  even  when  dilute.  The 
effect  of  protein-coagulating  antiseptics  upon  enzymes  is,  of  course,  greatly  modified 
by  the  amount  of  protein  substances  mingled  with  the  enzymes ;  and  the  effects  of 
heat  and  other  injurious  influences  are  greatly  decreased  by  the  presence  of 
proteins  and  other  impurities. 

All  enzymes  are  most  active  between  35°  and  45°  C,  and  it  is  interesting  to 
note  that  tvobert  found  this  equally  true  for  enzymes  derived  from  cold-blooded 
animals.'"  Although  enzymes  can  stand  temperatures  of  100°  C.  or  more  when 
dry,  in  water  they  are  generally  destroyed  somewhat  below  70°  C.  Low  tempera- 
ture, even  — 190°  C.  (liquid  air),  does  not  destroy  them.  The  loss  of  power 
through  heating  occurs  gradiially,  and  there  is  no  sharp  line  at  which  their  action 
disappears.  Sunlight  is  harmful  to  enzymes  in  solution,  but  only  in  the  presence 
of  oxygen;  this  effect  is  augmented  by  the  presence  of  fluorescent  substances. 
Nascent  oxygen  is  destructive  to  enzymes. '-'  Radium  and  .x-rays  seem  to  have  a 
deleterious  effect  upon  most  enzymes,  and  retard  their  rate  of  action ;  but  apparently, 
autolytic  enzymes  (Neuberg"-)  and  tyrosinase  (Willcock-'O  are  not  injured  by 
these  agencies. -^  Ultra  violet  rays  are  also  injurious  to  enzymes,-^  and  they 
can  be  destroyed  by  violent  shaking  (Shaklee  and  Meltzer.-*^).  Labile  as  enzymes 
are,  their  persistence  when  dry  is  remarkable;  Kobert  found  active  trypsin  in  the 
bodies  of  spiders  that  had  been  in  the  Nuremberg  Museum  for  150  years,  and  Seiirt'-^ 
found  that  the  muscle  tissue  of  mummies  contained  active  glvcolytic  ferment. 

All  enzymes  as  ordinarily  prepared  have  the  property  of  decomposing  hydro- 
gen peroxide,  a  property  possessed  by  substances  of  varied  nature;  this  effect  is 
prevented  by  CNH,  which  does  not  prevent  other  enzyme  manifestations,  indi- 
cating that  this  property  is  due  to  an  associated  enzyme,  catalase. 

The  retardation  of  enzyme  action  by  accumulation  of  the  products  of  their 

'9  See  Hedin,  Ergebnisse  d.  Physiol.,  1910  (9).  433. 

20  However,  Hosaka  (Mitt.  med.  Gesell.  Tokio,  1017  (31),  1)  states  that  frog 
pancreatic  diastase  is  most  active  between  5°  and  37°,  whereas  for  guinea  pig 
pancreatic  diastase  the  optimum  temperature  is  27°-55°.  Activity  begins  to  be 
inhibited  at  45°  and  65°  respectively. 

•^1  See  Burge,  Amer.  Jour.  Pliysiol.,  1914  (34),  140. 

"  Berl.  klin.  Woch.,  1904  (41),  1081. 

"  Jour,  of  Physiol.,  1906  (34),  207. 

24  Gudzent  (Zeit.  Strahlenther.,  1914  (4),  666)  denies  that  radium  acts  on 
enzymes. 

"  Agulhon,  Ann.  Inst.  Pasteur,  1912  (26),  38;  Burge  etal,  Amer.  Jour.  Phvsiol., 
1916  (40),  42C.. 

2«  Amer.  .lour.  Physiol.,  1909  (25),  81. 

"  Berl.  kliii.  Woch.,  1904  (41),  497. 


TOXICITY  or  ENZYMES  55 

action  is  simply  explained  as  being  due  to  establishment  of  equilibrium ;  in  some 
instances,  however,  the  substances  produced  are  of  themselves  harmful  to  the 
enzymes,  e.  g.,  alcohol  and  acetic  acid.  ('han^f'S  in  reaction,  fixation  of  the  enzyme 
by  cleavage  products,  and  other  side  reactions  may  also  be  at  least  partly  responsi- 
ble. There  is  a  periodicity  in  enzyme  action  which  makes  quantitative  results 
uncertain.'-'* 

Activation  of  Enzymes. — Within  the  cell,  the  enzymes — at  least  those  that  are 
excreted,  such  as  trypsin  and  pepsin — exist  with  few  exceptions  in  an  inactive 
form,  tiie  zymogen.  Their  activation  appears  to  take  place  normally  only  after 
they  have  been  discharged  from  the  cell,  but  after  the  death  of  an  organ  it  may 
result  from  the  decomposition  products  that  are  formed.  Under  physiological 
conditions  this  activation  appears  to  he  brought  about  by  special  activating 
substances.  In  the  case  of  the  pancreas  it  is  the  cnterokinase,  which  is  furnished 
by  the  epithelial  cells  of  the  intestine.  Enterokinase  appears  to  unite  with 
tr3'psinogen  to  form  an  active  enzyme,  which  reminds  one  of  the  way  that  comple- 
ment and  the  intermediarj-  body  unite  to  form  hemolytic  and  bacteriolytic 
substances.-^  Kinnses,  having  the  same  action  as  enterokinase  upon  the  trypsinogen, 
are  found  in  various  tissues  and  organs,  but  generally  much  less  active  than  the 
enterokinase.  It  is  verj^  probable  that  it  is  through  this  mechanism  that  the 
rate  of  enzyme  action  is  modified,  and  perhaps  it  is  a  means  of  defense  of  the  body 
against  its  own  enzymes;  as  the  prozymes  are  more  resistant  to  harmful  agencies 
than  the  enzymes,  it  also  may  be  a  method  of  storage.  The  activity  of  various 
enzymes  is  greatly  increased  by  certain  more  or  less  specific  substances,  referred 
to  usually  as  "coenz^-mes;"  thus  bile-salts  act  as  co-enzymes  for  lipase  (Loevenhart). 

The  Toxicity  of  Enzymes 

Although  present  normally  in  greater  or  less  amounts  in  all  the 
cells  in  the  body,  when  artificially  isolated  and  injected  directly  into 
animals  nearly  all  enzymes  seem  to  be  extremely  toxic.  As  foreign 
proteins,  especially  extracts  of  tissues,  are  generally  more  or  less 
toxic,  it  is  difhcult  to  state  how  much  of  the  toxicity  of  a  given  enzyme- 
containing  solution  depends  on  the  enzj^me  and  how  much  on  the 
admixt  proteins.  The  following  statements  are  taken  at  the  face  value 
placed  on  them  hy  the  several  investigators  quoted,  and  are  subject  to 
discount  until  the  enzymes  have  been  isolated  and  investigated  in  a  'pure 
condition,  if  such  a  thing  shall  ever  become  possible. 

The  first  thorough  study  of  the  toxicity  of  enzymes  was  made  by 
Hildebrandt,^"  who  found  that  pepsin,  invertase,  diastase,  emulsin, 
mja-osin,  and  rennin  were  all  toxic.  The  symptoms  produced  in  dogs 
were  trembling,  uneasiness,  difficulty  in  walking,  and  finally  coma. 
The  anatomical  changes  observed  were:  numerous  hemorrhages 
throughout  the  body,  fatty  degeneration  of  the  liver  and  myocardium, 
renal  congestion,  and  numerous  thromboses.  Considerable  fever  re- 
sults, and  Mayer  considers  this  responsible  for  the  relative  harmless- 
ness  of  rennin,  the  action  of  which  is  impaired  above  40°.  That  these 
effects  are  due  to  the  enzymes  themselves  rather  than  to  contaminating 

=8  GroU,  Nederl.  Tijdschr.  v.  Geneesk.,  1918  (1),  1085. 

2^  BayUss  and  StarUng  (Jour,  of  Physiol.,  1905  (32),  129),  question  the  anal- 
ogy of  zymogen-kinase  combinations  to  complement-amboceptor  combination. 
Walker,  however,  finds  evidence  that  many  enzymes  consist  of  a  specific  ambo- 
ceptor and  a  non-specific  complement  or  kinase  (Jour,  of  Physiol.,  1906  (33), 
p.  xxi.) 

30  Virchow's  Archiv,  1890  (121),  1. 


56  ENZYMES 

bacteria  is  shown  bj^  Kionka  and  by  Achalme^^  who  obtained  similar 
results  with  enzymes  made  sterile  by  filtration  through  porcelain. 
"Wago^2  obtained  also  an  amyloid-like  degeneration  widely  spread  in 
animals  injected  with  filtered  solutions  of  commercial  trj'psin,  pan- 
creatin  and  amylopsin.  Achalme  found  that  such  sterile  prepara- 
tions of  pancreatic  juice  injected  subcutaneously  into  guinea-pigs 
produce  a  marked  local  pink  gelatinous  edema,  followed  by  gangrene; 
if  the  animal  dies,  the  blood  is  non-coagulable. 

Apparently  cells  of  nearly  all  types  can  be  destroyed  by  trypsin, 
which  may  cause  necrosis  in  one-fourth  hour;  however,  spermatozoa 
and  surface  epithelium  resist  strong  trypsin  solutions.  Intravenous 
injections  cause  death  with  lesions  in  the  heart  muscle  and  severe 
hemorrhages.  After  recovery  from  one  injection  of  trypsin  the  animal 
is  temporarily  somewhat  more  resistant  to  another  injection,  and  there 
are  other  resemblances  to  anaphylactic  intoxication  (Kirchheim^^). 
Fiquet^^  also  observed  that  trypsin  and  pepsin  rendered  the  blood 
incoagulable,  but  after  some  time  the  coagulability  of  the  blood  is 
increased  and  thrombosis  is  frequent.  Wells^^  found  that  pancreatic 
extracts  containing  very  active  trypsin  and  lipase,  injected  intraperi- 
toneally,  produced  an  acute  inflammatory  reaction,  but  no  fat  necrosis. 
Extracts  containing  active  lipase  and  inactive  trypsin  were  less  toxic, 
but  produced  fat  necrosis.  Extracts  of  liver  and  blood  serum,  rich 
in  lipase,  were  almost  without  effect  on  dogs  and  cats. 

Pa-pain  was  found  to  be  much  more  toxic  than  any  animal  enzyme, 
causing  violent  local  hemorrhagic  inflammation.  Schepilewsky'^ 
also  found  papain  much  more  toxic  than  rennin  and  pancreatin; 
repeated  injection  of  the  two  latter  caused  amyloidosis  in  rabbits. 
Active  immunity  does  not  follow  repeated  injections  of  papain. ^"^ 
Lombroso^^  found  that  inactive  pancreatic  juice  was  much  less  toxic 
than  the  activated,  showing  that  it  is  the  trypsin  that  is  the  important 
toxic  agent.  He  also  found  that  succus  entericus  in  doses  of  1  to  5  c.c. 
is  toxic,  but  not  lethal  for  dogs.  Pancreatic  lipase  is  hemolj'tic 
(Noguchi^^)  if  activated  by  fats,  which  suggests  that  when  this  enzj^me 
gets  into  the  blood  it  may  cause  hemolysis.  Urease  has  a  definite 
toxicity  because  it  decomposes  the  urea  in  the  blood  and  tissues, 
fatal  intoxication  from  NH3  poisoning  resulting.*"  Hildebrandt*' 
observed  that  enzymes  were  positively  chcmotactic,  but  it  is  probable 

"  Ann.  d.  I'lnst.  Pasteur,  1901  CIS),  737. 

32  Arch.  Int.  Med.,  1919  (23),  251. 

"  Arch.  exp.  Path.  u.  Pharm.,  1911  (66),  352;  1914  f78),  99;  1913  (74),  374. 

^'  Arch.  d.  M('d.  Exper.,  1899  (11),  145. 

36  Jour.  Med.  Research,  1903  (9),  92. 

'8  Cent.  f.  Bakt.,  1899  (25),  849. 

"  Stenitzer,  Biochem.  Zeit.,  1908  (9),  382. 

38  Abstract  in  Biochem.  Centralblatt,  1903  (1),  712. 

3»  Biochem.  Zeit.,  1907  (6),  185. 

"  See  Carnot  and  Gerard,  Compt.  Rend.  Acad.  Sci.,  1919  (169),  88. 

<»  Virchow's  Arch.,  1893  (131),  5. 


ANTI-ENZYMES  57 

that  the  prcxkicts  of  their  action  on  the  tissues  are  the  chief  chemo- 
tactic  agents. 

The  enzymes  that  are  secreted  into  the  gastro-intcstinal  tract 
seem  to  be  chiefly  destroyed,  but  part  is  ehminated  in  the  feces,  and 
part  that  is  absorbed  apparently  reappears  in  the  urine  in  very  small 
quantities. *2  Pepsin,  diastase,  and  rerinin  all  have  been  found  in- nor- 
mal urine;  but  trypsin  is  present  chiefly  as  trypsinogen,  especially 
abundant  after  a  meat  diet.*^  Pepsin  and  rennin  enter  the  urine  as 
the  zjmiogens,  in  quantities  in  proportion  to  the  amount  in  the  stom- 
ach, and  are  absent  in  gastric  carcinoma  (Fuld  and  Hirayama**). 
During  resolution  of  pneumonia,  leucocytic  protease  may  appear  in 
the  urine  (Bittorf*^).  Ferments  injected  subcutaneously  are  said 
seldom  to  be  eliminated  in  any  considerable  amounts  in  the  urine, 
but  Opie^^  has  demonstrated  the  presence  of  lipase  in  the  urine  in 
pancreatitis  with  fat  necrosis,  and  Wago^^  found  that  injected  trypsin 
is  excreted  rapidly  and  abundantly.  Hildebrandt  was  able  to  prove 
that  emulsin  remained  active  for  at  least  six  hours  after  it  was  injected 
into  animals  subcutaneously,  by  its  splitting  amj^gdahn  which  was  then 
injected,  the  CNH  hberated  by  the  cleavage  of  the  amygdalin  causing 
death. 

Anti-enzymes 

Injection  of  enzymes  into  animals  leads  to  the  appearance  of  sub- 
stance^  in  the  serum  of  the  animals  that  antagonize  the  action  of  the 
enzymes.*^  The  principles  involved  are  quite  the  same  as  in  the  im- 
munization of  animals  against  bacterial  toxins  or  against  foreign  proteins. 
This  seems  to  have  been  first  observed  by  Hildebrandt,  and  it  has 
been  taken  up  extensively  in  recent  years  in  the  study  of  the  problems  of 
immunity\  An  interesting  observation  that  was  made  rather  early 
in  these  studies  was  that  normal  blood-serum  possesses  a  marked 
resistance  against  the  action  of  proteolytic  enzymes,  not  being  at  all 
digested  by  dilutions  of  enzymes  that  will  rapidly  digest  a  serum  that 
has  been  heated.  This  property  seems  to  be  shared  by  egg-white*^ 
and  by  the  tissues  and  organs  of  the  body  (Levene  and  Stookey^"). 
The  anti-enzyme  action  is  easily  destroyed  by  heat  of  about  70°,  by 
the   action  of  dilute  acids,  and  even  by  prolonged  standing.     It  is 

^2  Falk  and  KoUeb,  Zeit.  klin.  Med.,  1909  (68),  156. 

"^v.  Schoenborn,  Zeit.  f.  Biol.,  1910  (53),  386. 

"  Berl.  klin.  Woch.,  1910  (47),  1062. 

«  Deut.  Arch.  kUn.  Med.,  1907  (91),  212. 

«  Johns  Hopkins  Hosp.  BuU.,  1902  (13),  117. 

"  Jour.  Immunol.,  1919  (4),  19. 

*8  According  to  Porter  (Quart.  Jour.  Exper.  Physiol.,  1910  (3),  375)  enzymes 
in  contact  mth  various  membranes  are  inactivated,  and  substances  appear  which 
are  strongly  inhibitive  to  the  enzymes;  it  is  possible  that  this  effect  depends 
largely  on  zymoids,  which  unite  -wath  the  substrate  and  deviate  the  enzymes. 

"  Sugimoto,  Arch.  exp.  Path.,  1913  (74),  14. 

"  Jour.  Medical  Research,  1903  (10),  217. 


58  ENZYMES 

exerted  not  only  against  the  secreted  proteolytic  enzymes,  pepsin  and 
trypsin,  but  also  against  the  intracellular  enzymes  of  various  organs. 
We  therefore  distinguish  between  normal  and  immune  anti-enzymes. 

It  seems  highly  probable  that  the  resistance  of  the  body  tissues  to 
digestion  by  their  own  enzymes  and  by  the  enzymes  of  one  another 
depends  in  some  way  upon  the  presence  of  anti-enzymes  in  the  cells 
and  tissue  fluids,  for  self-digestion  of  tissues  is  greatly  impeded  by 
serum. ^^  Weiland^^  h^g  demonstrated  that  certain  intestinal  worms 
contain  a  strong  antitrypsin,  to  wliich  he  attributes  their  ability  to 
live  bathed  in  pancreatic  juice  without  being  digested. ^^  Similar 
properties  have  been  ascribed  by  other  observers  to  the  cells  of  the 
mucosa  of  the  stomach^'*  and  intestine,  and  to  the  mucus  itself  (de 
Klug),^^  but  the  work  of  Bensley  and  Harvey^^  indicates  that  the 
absence  of  free  acid  in  the  gland  cells  and  lumen  is  perhaps  the  chief 
protection  of  the  stomach  from  pepsin,  Kirchheim^^  holds  that  the 
intestines  are  protected  less  by  anti-enzymes  than  by  rapid  absorption 
and  removal  of  the  enzymes,  which  are  really  not  present  in  any  con- 
siderable excess  in  the  intestinal  contents.  The  anti-enzjmies  seem 
only  to  inhibit  enzyme  action,  and  not  to  destroy  the  enzjane  itself.^* 
Normal  anti-enzymes  do  not  seem  to  be  at  all  specific,  according  to 
V.  Eisler;^^  that  is,  human  serum  is  no  more  resistant  to  human  tryp- 
sin than  is  pig  serum — ^indeed,  it  is  less  so.^" 

Cathcart^^  found  that  normal  antitrypsin  is  connected  with  the 
"albumin  fraction"  of  the  serum,  i.  e.,  the  fraction  precipitated  between 
half  and  full  saturation  with  ammonium  sulphate.  Globulins  do  not 
possess  this  action,  but  they  are  not  easily  digested.  Antitrypsin  is 
found  in  all  varieties  of  serum,  and  is  little  or  not  at  all  specific.  It  is 
destroyed  by  Q5-70°C.^^  for  one-half  hour,  but  retains  its  anti-en  zj^matic 
activity  after  drying,  and  is  equally  effective  against  all  sorts  of  pro- 
teins.    The  normal  anti-tryptic  activity  decreases  during  fasting  and 

"  Wells,  Jour.  Med.  Research,  1906  (10),  149. 

*'■*  Zeit.  f.  Biol.  1903  (44),  45;  see  also  Dastre  and  Stassano,  Compt.  Rend. 
Soc.  Biol.,  1903  (55),  130  and  254;  and  Hamill,  Jour,  of  Physiol.,  1900  (33),  479. 

^^  Burge  (Jour.  Parasitol.,  1915  (1),  179)  suggests  that  the  protection  of  para- 
sites, and  perhaps  of  the  alimentary  epithelium,  depends  on  the  active  oxidative 
properties  of  these  tissues  destroying  the  enzymes. 

6'  See  Blum  and  Fuld,  Zeit.  klin.  Med.,  1906  (58),  505;  Langenskiold,  Skand. 
Arch.  Physiol.,  1914  (31),  1. 

"  Arch,  internat.  d.  physiol.,  1907  (5),  297. 

"  Biological  Bulletin,  1912  (23),  225. 

"  Arch.  exp.  Path.  u.  Pharm.,  1912  (71),  1. 

68  Bayliss  and  Starling  (Jour,  of  Physiol.,  1905  (32),  129;  and  Meyer,  Biochem. 
Zeit.,  1909  (23),  68,  oppose  the  view  of  Delezenne  that  the  antitryptic  action  of 
the  blood  is  due  to  an  antikinase,  and  believe  the  antibody  acts  upon  trypsin. 

"  Ber.  d.  Wien.  Akad.,  1905  (104),  119. 

«"  This  is  contradicted  by  Glaessner,  Hofmeister's  Beitriige,  1903  (4),  79. 

8'  Jour,  of  Physiol.,  1904  (31),  497;  also  see  Kiimmerer  and  Aubry,  Biochem. 
Zeit.,  1913  (48),  247. 

*-  Unless  otherwise  specified,  all  temperatures  are  given'according  to  the  Centi- 
grade scale. 


ANTI-ENZYMES  59 

increases  during  digestion  (Rosenthal^^) ;  it  is  increased  during  preg- 
nancy"'* and  the  blood  of  the  fetus  shows  less  than  that  of  the  mother. 
Normal  antitrypsin  unites  with  trypsin  according  to  the  law  of  mul- 
tiple proportions  (Meyer)  and  the  reaction  is  not  reversible  (Ronfloni). 
It  is  found  in  the  urine,  and  in  infianunatory  exudates,  but  not  in 
normal  serous  fluids,  and  it  resists  putrefaction.  Normal  serum  does 
not  seem  to  inhibit  the  enzymes  which  act  upon  purines.  Fuld  and 
Spiro"*  found  that  the  natural  antirennin  of  normal  horse  serum  is  in 
the  pseudoglobulin  fraction.  Since  acids  destroy  the  anti-enzyme 
property  of  the  serum,  it  is  not  effective  against  pepsin-HCl  mixtures. 
Against  trypsin,  however,  it  is  very  effective.  Zunz^*^  states  that  nor- 
mal serum  acts  more  upon  enterokinase  than  upon  trypsin,  and  believes 
that  the  inhibition  depends  upon  colloids  which  modify  surface  ten- 
sion and  adhere  to  the  proteins.  Red  corpuscles  and  living  unicellular 
organisms,  including  bacteria,  are  likewise  resistant  to  trypsin,  and 
normal  serum  also  seems  to  contain  an  antirennin. ^^ 

Oppenheimer  and  Aron^*  consider  it  probable  that  the  re- 
sistance of  normal  serum  to  trypsin  digestion  depends  upon  the  con- 
figuration of  the  protein  molecules,  which  perhaps,  when  in  fresh, 
uninjured  condition,  present  no  suitable  surfaces  for  attack  by  the 
ferment.  Hedin  attributes  antitrj^ptic  action  to  adsorption  of  the 
enzyme  by  some  constituent  of  the  serum,  much  as  charcoal  inhibits 
tryptic  digestion. 

Fresh  and  inactivated  serum  will  prevent  pepsin  from  digesting 
protein,  but  this  is  not  due  to  a  true  antipepsin,  according  to  Ham- 
burger."^ 

Jobling^"  and  his  co-workers  have  advanced  evidence  that  the  nor- 
mal antiprotoase  action  of  serum  depends  on  the  lipoids  of  the  senuii,^^ 
which  var}^  in  activity  directly  with  the  degree  of  unsaturation;  there- 
fore they  were  able  to  decrease  the  antiferment  action  of  serum  by 
extracting  the  lipoids  with  fat  solvents  (and  to  restore  the  activity 
by  replacing  the  lipoids),  or  by  saturating  the  double  bonds  of  the 
fatty  acids  with  halogens,  or  by  modifying  the  degree  of  dispersion 

8'  Folia  Serologica,  1910  (6),  285;  also  Franz  and  Jarisch,  Wien.  klin.  Woch., 
1912  (25),  1441. 

"  See  Franz,  Arch.  f.  Gyn.,  1914  (102),  579. 

«5Zeit.  f.  physiol.  Chem.,  1900  (31),  132. 

*8  Mem.  Acad.  roy.  med.  Belgique,  1909  (20),  fasc.  5. 

"  Czapek  (Ber.  Deut.  botan.  Gesell.,  1903  (21),  229)  states  that  anti-oxidases 
occur  normally  in  certain  plants,  strongly  specific  against  the  oxidase  of  the  same 
plant  species. 

°8  Hofmeister's  Beitrage,  1903,  (4),  279. 

«3  Jour.  Exper.  Med.,  1911  (14).  535;  Arch.  Int.  Med.,  1915  (16),  356.  There 
seems  to  be  no  relation  between  the  antipeptic  and  antitrvptic  powers  of  sera 
(Rubinstein,  Ann.  Inst.  Pasteur.,  1913  (27),  1074). 

I  1  '"  Series  of  articles  in  Jour.  Exper.  Aled. ;  also  review  in  Jour.  Lab.  and  Clin. 
Med.,  1915  (1),  172.     See  also  Zeit.  Immunitat.,  1914  (23),  71. 

"  Yamakawa  (Jour.  Exp.  Med.,  1918  (27),  689),  liowever,  does  not  believe 
that  the  antienzyme  which  prevents  autolysis  of  serum  itself  is  of  lipoidal  nature. 


60  ENZYMES 

of  the  lipoids.     Soaps  of  saturated  fatty  acids  do  not  inhibit  serum 
protease. 

Opie^2  has  found  that  the  serum  of  inflammatory  exudates  con- 
tains an  anti-enzymatic  substance,  destroyed  at  75°  and  by  acids; 
it  is  not  present  in  normal  cerebrospinal  fluid,  but  appears  here  as 
in  other  serous  cavities  during- inflammation  (Dochez).''^  Antitrypsin 
has  also  been  found  in  pathological  urines  (v.  Schoenborn).^'* 

The  power  of  the  blood  serum  to  inhibit  the  activity  of  trypsin  and 
leucocytic  protease  has  been  found  to  vary  greatly  in  disease,  and,  as 
having  diagnostic  possibilities,  this  property  has  been  considerably 
investigated.''^  It  is  especially  increased  in  conditions  associated  with 
cell  destruction,  such  as  pneumonia  and  cancer,  which  suggests  that 
the  increased  antitryptic  activity  results  from  the  formation  of  specific 
antibodies  for  the  intracellular  proteases  liberated  during  the  disease, 
but  as  yet  this  has  not  been  satisfactorily  established,  so  we  do  not 
know  whether  the  "antitrypsin  reaction"  depends  upon  an  antibody 
for  trypsin  or  upon  some  entirely  different  factor.  In  cachexia  the 
inhibiting  effect  of  the  serum  is  especially  marked  and  it  is  therefore 
usually  pronounced  in  cancer,  but  the  increased  inhibition  is  some- 
times absent  in  cancer  (10  per  cent,  of  all  cases)  and  often  present  in 
other  conditions,  so  that  the  positive  diagnostic  value  is  slight.  It 
may  also  be  present  without  cachexia  and  often  seems  to  parallel  the 
number  of  leucocytes  in  the  circulating  blood.  Sarcoma  shows  it 
less  than  carcinoma,  while  in  exophthalmic  goitre  and  tuberculosis 
an  antitryptic  increase  is  said  to  be  quite  constant  (Waelli).''®  In 
pregnancy  there  is  usually  an  increase  demonstrable  after  the  fourth 
to  sixth  months,  continuing  until  two  weeks  after  delivery,  and  highest 
in  cases  of  pregnancy  toxemias  (Ecalle)."  Severe  traumatism  may 
also  cause  an  increase.''^ 

As  normal  serum  contains  a  tryptic  enzyme  as  well  as  a  substance 
inhibiting  trypsin,  the  antitryptic  activity  is  at  most  but  a  measure  of 
the  difference  between  these  (Weil),  and  might  depend  on  either 
lowered  trypsin  or  increased  antitrypsin  content.  Doblin^^  and 
many  others  believe  with  Jobling  that  the  active  agent  is  not  a  true 
immune  antibody,  but  as  yet  general  agreement  has  not  been  reached 
on  this  point  (see  Meyer).  Kirchheim*"  has  found  that  the  union  of 
trypsin  and  antitrypsin  does  not  follow  the  physico-chemical  laws  to  a 
true  antigen-antibody  reaction.     Rosenthal  has  advanced  evidence 

^2  Jour.  Exp.  Med.;  1905  (7),  316. 
"Jour.  Expcr.  Med.,  1909  (11),  718. 
''*  Zeit.  f.  Biol,  1910  (53),  386. 

^^  For    literature  and  review  see  Wiens,   Ergebnisse   Phj'siol.,    1911    (15),    1; 
Weil,  Arch.  Int.  Med.,  1910  (5),  109;  Meyer,  Folia  Serologica,  1911  (7),  471. 
'«  Mitt.  Grenz.  Med.  u.  Chir.,  1912  (25),  184. 
"  Arch.  Mens.  Obst.  Gvn.,  1917  (6),  97. 
"  Zunz  and  Govaerts,  C.  R.  Soc.  Biol.,  1918  (81),  146. 
'»  Zeit.  f.  Immunitilt,  1909  (4),  229 
80  Arch.  exp.  Path.,  1913  (73),  139. 


ANTI-ENZYMES  61 

to  support  the  hypothesis  that  the  presence  of  ])ro(hicts  of  protein 
cleavage  in  the  serum  is  responsible  for  the  antitryptic  action,  but  this 
has  not  been  confirmed.  Attempts  have  been  made  to  regulate  sup- 
purative processes  by  the  introduction  of  either  leucocytic  proteases, 
or  antiprotease  in  the  form  of  active  serum  (see  Wiens^^).  Whether 
antiprotease  can  be  specifically  developed  by  immunizing  with  leuco- 
protease  is  a  matter  of  disagreement,^^  but  no  increase  of  antiprotease 
follows  the  enormous  destruction  of  leucocytes  caused  by  injecting 
thorium- A'.  ^2 

The  anti-enzymatic  property  obtained  in  the  serum  by  injecting 
enzj'mes  into  animals  differs  from  that  normall}'"  present  in  the  serum 
in  many  ways.  It  may  be  made  much  stronger  than  it  ever  is  in 
normal  serum,  and  against  many  varieties  of  enzymes  for  which  an 
anti-enzjaiie  does  not  naturally  exist.  Especiall}^  important  is  the 
fact  that  it  is  highly  specific  (v.  Eisler);  serum  of  an  animal  immu- 
nized against  dog  trypsin  will  show  a  much  greater  ef5"ect  against  dog 
trypsin  than  it  does  against  trypsin  from  other  animals.  This  fact 
permits  us  to  distinguish  between  enzymes  of  apparently  similar 
nature  but  of  different  origin,  and  proves  that  they  have  a  struc- 
ture at  least  in  some  respects  different  from  one  another,  since  they 
are  combined  by  different  antibodies.  Apparently  that  element  of 
the  enzymes  which  determines  their  action  on  specific  substances  is 
involved  in  their  antigenic  properties,  since  antiproteases  will  not 
inhibit  diastase  or  lipase.  This  specificity  is  limited,  however,  for 
the  anti-enzymes  for  leucocytic  and  pancreatic  proteases  are  said  to  be 
identical. ^^  Artificial  immune  serum  is  said  to  have  been  obtained 
against  trj^psin,  pepsin, ^^  lipase,  emulsin,^'^  autoh-tic  enzymes,  laccase, 
amylase,  invertin,  diastase,  tjTOsinase,  urease,^^  rennin,  catalase,  and 
fibrin  ferment. ^^  By  immunization  against  bacteria  an  immunity 
against  their  proteolytic  enzymes  is  also  obtained, ^^  which  is  inde- 
pendent of  and  different  from  antitr3^psin,  being  especially  in  the 
globulin  fraction,  while  the  antibody  for  pancreatic  trypsin  is  chiefly 
in  the  albumin  (Kammerer^^).  From  the  work  of  Kirchheim  and 
Reinicke^^  it  seems  probable  that  the  increased  resistance  following 

81  See  Bradley,  Jour.  Hyg.,  1910  (12),  209. 

*-  G.  Rosenow  and  Farber,  Zeit.  exp.  Med.,  1914  (3),  377. 

8'  Jochmann  and  Kantorowicz,  Mlinch.  med.  "Woch.,  1908  (55),  728. 

«^  Bayliss  (Jour,  of  Physiol.,  1912  (43),  455)  was  unable  to  obtain  antiemulsin, 
and  Pozerski  (Ann.  Inst.  Pasteur,  1909  (23),  205)  failed  to  obtain  antipapain, 
but  positive  results  are  reported  by  v.  Stenitzer  (Biochem.  Zeit..  1908  (9),  382). 

**  Jacoby  says  that  the  disappearance  of  urease  from  the  blood  after  repeated 
injection  does  not  depend  on  the  formation  of  an  antienzyme  (Biochem.  Zeit., 
1916  (74),  97). 

8*  For  a  review  of  much  of  the  earlier  literature  on  this  subject  see  Schiitze, 
Deut.  med.  Woch.,  1904  (30),  308. 

8'  Dungern,  Miinch.  med.  Wochenschr.,  1898  (45),  1040;  Bertiau,  Cent,  f . 
Bact.,  1914  (74),  374. 

s8  Deut.  .\rch.  klin.  Med.,  1911  (103),  341. 

«9  Arch.  exp.  Path.,  1914  (77),  412. 


62  ENZYMES 

immunization  with  trypsin  is  simply  an  increase  in  nonspecific  resist- 
ance, such  as  follows  injection  of  peptone  and  man}'  other  poisonous 
substances.  Wago*^  was  able  to  demonstrate  precipitins  and  com- 
plement fixing  antibodies  in  antitryptic  sera  that  were  not  strongly 
antienzymatic,  and  Young^°  was  unable  to  produce  antitryptic 
sera  by  immunizing  with  trypsin-,  in  spite  of  the  presence  of  active 
precipitins  for  the  injected  trypsin  solutions.  There  is,  indeed,  a 
growing  suspicion  that  much  of  the  evidence  of  specific  antibodj^ 
formation  for  enzymes  must  be  revised. 

Resemblances  of  Enzymes  and  Toxins. — As  can  be  seen  from  the  al)ove  state- 
ments, the  enzymes  behave  in  many  respects  lilce  the  toxins,  both  in  tlieir  manner 
of  acting  upon  other  substances  and  in  the  reaction  they  produce  when  introduced 
into  the  bodies  of  animals.  As  Oppenheimer  says,  "the  bonds  between  enzymes 
and  toxins  are  drawing  closer  and  closer."  According  to  some  experiments,  the 
enzymes  behave  much  as  if  they  possessed  a  haptophore  and  a  toxophore  group, 
the  former  of  which  combines  with  the  substance  that  is  to  be  acted  upon;  and 
immunity  appears  to  be  produced  by  the  development  of  receptors  that  combine 
the  haptophore  groups,  these  receptors  constituting  the  antiferments.  There  is 
abundant  evidence  of  a  toxin-like  structure  in  enzymes,  from  the  numerous  ob- 
servations on  the  formation  of  "zymoids"  which  can  neutralize  anti-enzymes 
or  combine  with  the  substrate,  although  no  longer  active  as  enzymes.  The 
oxidizing  enzymes  especially,  with  their  complex  relationship  of  substrate,  com- 
bining body  (peroxides)  and  enzyme,  present  striking  analogies  to  immune  reac- 
tions (Moore^')?  and  the  proteolytic  substances  of  the  blood  resemble  the  lysins 
in  certain  respects  (Dick).'^  Enzymes  and  toxins  also  resemble  one  another  in 
being  readily  absorbed  by  membranes,  precipitates,  and  highly  developed  sur- 
faces in  general.^'  Finally,  there  is  much  reason  to  believe  that  the  hemolytic 
toxin  of  cobra  venom  is  a  lipase,  which  acts  by  splitting  lecithin  into  hemolytic 
substances  (Coca).^^ 

THE  INTRACELLULAR  ENZYMES  « 

Until  a  recent  time  our  knowledge  of  enzymes  in  the  animal  body 
was  limited  to  those  present  in  the  digestive  secretions.  With  few 
exceptions  these  are  without  influence  in  pathological  processes,  since 
they  seem  to  be  but  little  absorbed,  and  rarely  enter  the  blood  or 
tissues  in  any  other  way.  But  with  the  more  recently  disclosed  intra- 
cellular enzymes,  many  of  which  are  present  in  every  cell,^^  the  rela- 
tion to  pathology  is  very  intimate.  These  intracellular  enzymes,  as 
we  now  know  them,  and  their  chief  properties,  are  as  follows: 


s"  Biochem.  Jour.,  1918  (12),  499. 

9'  Biochem.  Jour.,  1909  (4),  165. 

»2  Jour.  Infectious  Diseases,  1911  (9),  282. 

9'  See  Porter,  C,)uart.  Jour.  Exp.  Physiol.,  1910  (3),  375. 

9' Jour.  Infect.  Dis.,  1915  fl7),  351. 

^'' Sv.G  Vernon,  Ergebnissc  d.  Pliysiol.,  1910  (9),  138;  also  liis  monograph, 
"Intracellular  Enzvmes,"  London,  1908. 

9«  Hcrlitzka  (Arch.  ital.  biol.,  1907  (48),  119)  and  others  have  shown  that 
the  diiferent  enzymes  appear  one  by  one  in  the  development  of  tiie  ovum.  Their 
activity  is  modified  considerably  by  infections  (Siel)er,  Hiochem.  Zeit.,  1911  (32), 
108)  aiid  other  diseases  ((irossinanh,  ibid.,  1912  (11),  ISl). 


OXIDIZING  ENZYMES  ()3 


OXIDIZING  ENZYMES" 


Although  oxidation  of  organic  compounds  is  the  chief  sour(;c  of 
energy  in  the  animal  body,  yet  the  way  in  which  it  is  accomplished 
is  very  little  understood.  We  only  know  that  it  is  brought  about 
within  the  cells,  and  that  substances  that  outside  the  body  are  oxidized 
with  difficulty,  are  completely  oxidized  to  car})on  dioxide  and  water 
within  the  cells,  and  that  this  is  done  with  just  such  a  degree  of  rapid- 
ity that  the  heat  produced  is  in  exactly  the  amount  necessary  for 
the  wants  of  the  bod3^  There  can  be  little  question  that  this  oxida- 
tion is  accomplished  through  catalytic  agents  acting  within  the  cells, 
and  certain  of  them  have  been  placed  in  a  condition  permitting  of 
study.  As  yet  their  exact  relations  to  intracellular  oxidation  are 
not  clearly  defined,  but  for  the  present  they  may  be  grouped  pro- 
visionally as  oxidizing  enzymes.  That  some  of  them  arc  highly  specific 
is  shown  by  those  disorders,  such  as  alkaptonuria  and  diabetes,  in 
which  the  body  loses  the  power  to  oxidize  a  certain  chemical  substance 
while  retaining  the  normal  power  to  oxidize  innumerable  other  sub- 
stances. According  to  Lillie^^  the  oxidative  processes  in  cells  take 
place  most  actively  in  relation  to  the  membrane  surfaces  (or  phase 
boundaries)  of  the  cells.  Of  the  oxidizing  enzymes  as  yet  identified 
none  can  be  considered  as  of  importance  in  the  energy-producing 
oxidations  of  the  body  (Battelli  and  Stern),  all  the  enzymes  of  this 
class  yet  known  being  apparently  concerned  with  less  essential  oxidiz- 
ing processes;  it  is  indeed  possible  that  the  essential  oxidation  of 
food-stuffs  ma}^  not  be  dependent  on  enz3'mes  (Engler  and  Herzog).^^ 
An  agent  accelerating  the  essential  oxidizing  activities  of  the  tissues 
has  been  described  by  Battelli  and  Stern'  under  the  name  of  pnein,  and 
an  anti-pneumin  which  holds  it  in  check.  Closely  related  to  the  oxidiz- 
ing enzj^mes  is — • 

Catalase. — It  has  long  been  known  that  most  enzymes  possess 
the  power  of  decomposing  hydrogen  peroxide,  with  liberation  of 
oxygen;  but  it  was  not  until  1901  that  it  was  finally  demonstrated  by 
Loew  that  this  property  was  due  to  a  separate  enzyme  and  was  inde- 
pendent of  the  specific  properties  of  the  various  other  enzymes.  This 
ferment  is  very  wide-spread,  and  so  is  generally  obtained  along  with 
the  other  enzymes  when  attempts  are  made  to  isolate  them  from  the 
cell.     It  was  named  catalase  by  Loew,  and  he  described  two  forms,  a- 

9^  Complete  bibliography  and  exhaustive  discussion  by  Kastle,  Bull.  Hygienic 
Lab.,- No.  59;  by  Loele,  Ergeb.  allg.  Path.,  1912  (16,  Pt.  2),  760;  and  by  BattelU 
and  Stern,  Ergebnisse  d.  Phj-siol.,  1912  (12),  96.  Concerning  the  chemistry  of 
vital  oxidations  see  Dakin,  "Oxidations  and  Reductions  in  the  Animal  Body," 
Monographs  on  Biochemistry,  London,  1912.  Good  review  by  v.  Fiirth.  "Chem- 
istry of  Metabolism,"  Chaps.  22  and  23;  translated  by  A.  J.  Smith,  Philadelphia, 
1916. 

38  Jour.  Biol.  Chem.,  1913  (15),  237. 

93  Zeit.  phvsiol.  Chem.,  1909  ^59),  327. 
iBiochem.  Zeit.,  1911  (33),  315;  1911  (36),  114. 


64  ENZYMES 

catalase,  which  was  thought  to  be  a  nucleoprotein,'^  and  ^-catalase, 
which  has  the  properties  of  an  albumose.  It  has  been  demonstrated 
by  Bach  and  Chodat  that  peroxides  are  contained  in  plant  cells,  and 
they  also  occur  in  animal  cells.  According  to  Golodetz  and  Unna^ 
the  catalases  are  held  in  the  cytoplasm  of  the  cells  while  the  peroxida- 
ses are  in  the  nucleus.  Just  what  function  the  catalase  performs  is  at 
present  merely  a  matter  of  speculation,  but  that  it  serves  an  important 
purpose  is  indicated  by  the  observation  of  Burge*  that  the  amount  of 
catalase  in  tissues  varies  directly  with  their  activity.  He  also  as- 
cribes the  specific  dynamic  action  of  proteins  to  their  causing  an 
increase  in  blood  catalase.  Becht,^  however,  questions  the  validity 
of  the  evidence  so  far  brought  forward  in  support  of  the  hypothesis 
that  catalase  is  essentilly  responsible  for  tissue  oxidation.^ 

Loew  considers  that  it  destroys  peroxides  formed  in  metabolism, 
which|are]very  poisonous  to  cell  life.  Shaffer  has  found  evidence  that 
under  the  influence  of  catalase  the  oxygen  liberated  is  in  the  molecular 
form,  O2,  and  therefore  relatively  inert;  whereas  when  peroxides 
spontaneously  decompose,  they  liberate  atomic  oxygen  which  is  an 
active  oxidizing  agent.  He  found  that  uric  acid  is  oxidized  by  per- 
oxide of  hydrogen,  but  when  catalase  is  present,  this  oxidation  is 
prevented.  According  to  this  the  function  of  catalase  is  rather  to 
prevent  dangerous  forms  of  oxidation  than  to  help  in  normal  oxi- 
dative proceses.  For  the  present,  however,  nothing  can  be  said 
positively  on  this  subject. 

Occurrence  of  Catalase  under  Normal  and  Pathological  Conditions.'' — Battelli  and 
Stern  found  that  the  catalytic  power  of  the  tissues  endures  many  hours  after 
death.  Its  abundance  is  different  for  different  organs  of  the  same  animal,  but 
remarkably  constant  for  the  same  organ  in  the  same  species.  In  general  the  order 
in'^decreasing  strength  is:  liver,  kidney,  blood,  spleen,  gastro-intestinal  mucosa, 
salivary  glands,  lung,  pancreas,  testicle,  heart,  muscle,  brain;  but  this  order  varies 
in  different  species.  Catalase  is  abundant  even  in  the  early  embryo  (^lendel  and 
Leavenworth)  and  in  sea  urchin  eggs  it  increases  rapidly  after  they  are  fertilized 
(Lyon). 8  Leucocytes  contain  little,  most  of  that  in  the  blood  being  in  the  stroma 
of  the  red  blood-corpuscles.  The  body  fluids  contain  little  or  none.  Injected 
intravenously,  catalase  (of  the  liver)  is  destroyed  rapidly,  and  docs  not  appear  in 
the'-urine;  it  does  not  cause  any  toxic  effects,  nor  does  it  increase  resistance  to 
poisoning  by  venoms.  The  tissues  also  contain  anti-catalases,  and  still  further  a 
substance  which  protects  the  catalase  froni  the  anti-catalase;  this  protective  sub- 
stance is  called  the  pliilocatalase  by  Battelli  and  Stern. 

The  gas  evolved  by  the  action  of  pus  on  H2O2  was  found  by  Marshall'  to  be  pure 
oxygen,  each  c.c.  of  a  certain  sample  of  pus  examined  liberating  133.9  c.c.  of  gas. 
The  active  constituent  of  pus,  he  states,  is  contained  in  the  serum  and  not  in  the 

2  Not  corroborated  by  Waentig  and  Gierisch,  Fermentforsch.,  1914  (1;,  165. 

'  Berl.  klin.  Woch.,  1912  (49),  1134. 

•'  vVmer.  Jour.  I'liysiol.,  191G  (41),  153;  1917  (42),  373;  1919  (48).  133.  See  also 
Alvarez  and  Starkweather,  ibid.;  1918  (47),  GO;  Dutcher,  Jour.  Biol.  Chem.,  1918 
(36),  63. 

5  Amer.  Jour.  Physiol.,  1919  (48),  171. 

8  See  also  Stelilc,  Jour.  Biol.  Chem.,  1919  (39),  403. 

^  Concerning  the  catalase  of  lower  animals  see  Ziegcr,  Biochem.  Zeit.,  1915 
(69),  39. 

8  Amer.  Jour.  Physiol.,  1909  (25),  199. 

»  Univ.  of  Pcnn.  Med.  Bull.,  1902  (15),  366. 


OXIDIZING  ENZYMES  65 

corpuscles.  Catalase  is  abundant  in  the  tissues  of  lower  animal  forms,  e.  g., 
Ascaris.^"  Substances  decomposinp;  HoOj  have  been  found  also  in  bacterial  cul- 
tures, first  by  Gottstein,  and  later  in  the  cell  juices  expressed  from  tubercle  bacilli 
by  Hahn.  Locwenstein"  found  an  enzyme  aRreeinp;  with  catalase  in  filtered  bouillon 
cultures  of  diphtheria  bacilli  and  staphylococci,  l)ut  not  frorn  tetanus,  typhoid, 
and  colon  bacilli  or  cholera  vibrios;  the  catalase  is  quite  distinct  from  the  toxin. 
He  also  found  that  the  addition  of  H2O2  to  a  diphtheria  toxin-antitoxin  mixture 
destroyed  the  toxin,  leaving  the  antitoxin  free.  A  similar  destruction  of  tetanus 
toxin  l)y  peroxides,  first  demonstrated  by  Sieber,  can  occur  without  the  catalase. 

Winternitz^-  and  his  associates  have  made  extensive  studies  of  the  catalase 
activity  of  the  blood  and  tissues  in  disease.  They  found  that  all  tissues  have  re- 
duced catalase  activity  in  chronic  nephritis,  in  proportion  to  the  severity  of  the 
condition,  and  experimental  nephritis  in  animals  has  the  same  effect;  the  blood 
shows  great  reduction  in  catalase  in  vu-emia,  and  a  less  reduction  with  less  severe 
nephritic  manifestations.  Eclampsia  shows  little  or  much  reduction  of  catalase 
in  the  blood  in  proportion  to  the  amount  of  renal  involvement;  normal  pregnancy 
and  labor  have  no  elTect.  Anemia  is  associated  with  irregular  decrease  in  catalase, 
including  primary  anemias  and  the  secondary  anemias  of  typhoid  and  pneumonia; 
cardiac  disease  has  no  effect  if  the  kidneys  are  normal.  Acute  peritonitis  causes  a 
rise  in  blood  catalase;  diabetes,  leukemia  and  jaundice  were  w-ithout  effect.  In 
hyperthj-reosis  the  catalase  tends  to  increase,  in  hypothyreosis  to  decrease;  com- 
plete removal  of  the  thyroid  causes  a  decrease  which  disappears  on  feeding  thy- 
roid. Intravenous  injection  of  salts,  acids  and  alkalies  decreases  the  catalytic 
activity  of  the  blood.  In  shock,  blood  catalase  is  decreased."  Normal  indi- 
viduals show  considerable  variations  in  the  catalase  activity  of  the  blood,  but  for 
each  individual  it  is  remarkably  constant;  age  has  very  little  influence.  In  the 
tissues  post  mortem  change  causes  but  slight  reduction  in  catalase.  Extirpation  of 
large  amounts  of  kidney  or  liver  tissue  has  little  effect,  but  removal  of  the  spleen, 
ovaries  or  testicles  causes  a  transient  decrease  in  the  catalase  of  the  blood.  In 
diabetes  and  starvation,  tissue  catalase  is  said  to  be  decreased.'^  If  the  red  cor- 
puscles are  prevented  from  laking,  the  catalase  activity  manifested  by  the  blood  in 
vitro  is  reduced  (Strauss)'*  and  iodides  increase  the  catalase  activity  of  the  blood. 
Catalase  and  anticatalase  have  been  found  in  pathological  urine,  in  both  acute 
and  chronic  nephritis  (Primavera).'^  Kahn  and  Brim'^  also  found  traces  of  cata- 
lase in  normal  urine,  greatly  increased  in  urine  containing  blood,  bile  or  acetone, 
normal  in  cancer,  high  in  diabetic  acidosis,  Hodgkin's  disease,  septic  infections  and 
typhoid.  Grossman'*  foimd  that  bacterial  poisons  generally  increase  the  catalase 
content  of  the  various  viscera,  and  Rosenthal'^  observed  a  great  decrease  in  the 
liver  and  blood  of  mice  receiving  intraperitoneal  inoculations  of  cancer.  The 
catalase  activity  of  the  non-cancerous  organs  of  cancer  patients  is  not  affected, 
except  slightly  lowered  by  cachexia  (Colwell) ;'°  however,  the  liver  tissue  between 
cancer  nodules  may  show  less  catalase  than  normal  liver.  •^'  In  phosphorus  poi- 
soning the  catalase  content  of  the  liver,  heart  and  blood  is  decreased  (Burge).'-' 

But  it  is  to  be  borne  in  mind  that  the  questionable  accuracy  of  our  existing 
methods  of  determining  quantitatively  the  amount  or  activity  of  catalase  in  tis- 
sues makes  the  foregoing  statements  of  uncertain  value. 

True  Oxidizing  Enzymes. — While  it  is  by  no  means  certain  that 
catalase  is  active  in  causing  intracellular  oxidations,  there  are  other 

'0  Magath,  Jour.  Biol.  Chem.  1918  (33),  395. 

"  Wien.  klin.  Woch.,  1903  (16),  1393. 

12  Review  in  Arch.  Int.  Med.,  1911  (7),  624. 

»3  Burge  and  Neill,  .\mer.  Jour.  Physiol.,  1918  (45),  286. 

1*  Burge,  Science,  1918  (47),  347. 

'6  Bull.  Johns  Hopkins  Hosp.,  1912  (23),  120. 

'6  Riforma  Med.,  1906  (12),  1266. 

"  Amer.  Jour.  Obst.,  1915  (71),  39. 

18  Biochem.  Zeit.,  1912  (41),  181. 

19  Deut.  med.  Woch.,  1912  (38),  2270. 

20  Arch.  Middlesex  Hosp.,  1910  (19),  64. 

21  Blumenthal  and  Brahn,  Zeit.  Krebsforsch.,  1910  (8),  436. 
"  Amer.  Jour.  Phvsiol.,  1917  (43),  545. 


66  ENZYMES 

enzymes  or  enzyme-like  substances  that  come  more  properly  under  the 
head  of  oxidases  or  oxidizing  enzymes.  Battelli  and  Stern  contend 
that  the  only  real  oxidases  which  have  yet  been  completely  established 
are:  1.  Polyphenoloxidases  (oxidizing  phenols  and  their  amino  com- 
pounds, but  not  tyrosine);  2.  Tyrosinase;  3.  Alcohol  oxidase;  4. 
Xanthine  oxidase;  5.  Uricase.  Chodat  and  Bach  believe  that  the 
enzymes  which  are  designated  above  as  polyphenoloxidases  have  a  com- 
plex structure,  consisting  of  peroxidase  and  oxygenase. ^^  Mathews^* 
holds  that  "under  the  term  oxidases  there  have  been  confused  two 
classes  of  substances,  one  which  activates  the  oxygen;  the  other  the 
more  important  class,  which  activates,  by  dissociation,  the  reducing 
substances.  The  latter  are  specific,  the  former  not."  This  view  has 
received  support  by  Bach. 

Peroxidase. — This  name  is  given  to  an  enzyme  that  is  believed  to  cause  oxida- 
tion by  activating  peroxides,  and  is  quite  distinct  from  catalase  and  from  the  other 
oxidases.  The  peroxide  on  wluch  it  chiefly  acts  in  the  cell  is  supposed  to  be  the 
so-called  "oxygenase." 

Oxygenase. — This  can  also  act  as  an  oxidizer  independent  of  the  peroxidase, 
in  the  presence  of  certain  manganese  compounds.  Loevenhart  and  Kastle  ques- 
tion the  true  enzyme  nature  of  this  and  other  "oxidases,"  which  they  look  upon  as 
organic  peroxides,  behaving  like  other  peroxides  rather  than  as  catalyzers.  Prac- 
tically tlie  existence  of  these  bodies  is  demonstrated  by  their  power  to  turn  tinc- 
ture of  guaiac  blue,  and  they  are,  therefore,  present  in  pus. 

Von  Fiirth^^  sums  up  the  situation  in  these  words:  "In  the  tissues  active  cata- 
lytic agents,  the  peroxidases,  are  widely  distributed;  which  seem,  just  like  the 
coloring  matter  of  the  blood,  to  be  capable  of  conveying  the  ox3^gen  from  peroxides 
to  very  readily  oxidizable  substances.  We  find  too  in  the  statements  bearing 
upon  the  oxygenases,  the  aldehydases  and  indophenoloxidases,  occasion  for  assiun- 
ing  that  there  are  substances  in  the  tissues  charged  with  oxygen  which  are  able 
to  give  this  off  to  easily  oxidizable  matter;  and  these  we  may  in  a  measure  regard 
as  peroxides.  But  that  is  all.  We  do  not  know  whether  the  peroxidases  are 
ferments  or  not." 

By  their  conception  of  oxygenase  and  peroxidase  Chodat  and  Bach  would 
displace  entirely  the  idea  of  enzymes  oxidizing  directly,  the  true  "oxidases,"  which 
they  consider  mixtures  of  oxygenase  and  peroxidase.  There  have  been,  in  any 
event,  a  number  of  ferments  described  that  seem  to  possess  distinct  oxidative 
powers.  As  each  is  quite  specific  in  its  action,  oxidizing  but  one  substance,  or  one 
group  of  related  substances,  they  are  generally  designated  by  the  name  of  the 
substances  upon  which  thej^  act.  Most  studied  of  these  are  aUlehytlase  and 
tyrosinase. 

Aldehydase,^^  which  is  characterized  l)y  oxiilizing  aldehydes,  particularly 
salicyl-aldehydc.  According  to  Jacquct,  this  enzyme  is  so  intinuitely  bouutl  with 
the  cell  that  it  cannot  l)e  olitained  in  extracts  until  after  the  cells  are  dead,  but  is 
present  in  expressed  cell-juices.  It  can  be  isolated  by  the  usual  metliods,  is  de- 
stroyed by  boiling,  acts  best  when  no  free  oxygen  is  present,  and  its  action  is  in- 
hibited 1j3'  CNH.  It  lias  been  demonstrated  in  nearly  all  organs  and  tissues  except 
pancreas,  nuiscle,  marrow,  and  mammary  gland;  it  is  present  in  the  blood  in  small 
amounts,  but  not  at  all  in  the  bile.  It  is  most  abundant  in  the  liver-*^  and  spleen, 
and  is  present  in  jjig  embryos,  9  cm.  long,  but  not  in  tliose  2-3  cm.  long.  Jacoby 
has  obtained  a  body  with  the  properties  of  aldehydase  which  did  not  give  protein 
reactions.     It  is  a  true  enzyme,  since  it  o.xidizes  aldeliydes  without  itself  being 

^' See  also  Onslow,  Biochcm.  Jour.  l'.)l'.)  (13),  1. 
^-i  Jour.  Hiol.  Chem.,  190<J  ((>),  1. 

■■'^  Battelli  and  Stern  do  not  include  aldeliydase  among  the  oxidizing  enzymes, 
on  the  ground  tliat  its  action  is  not  oxidative  but  liydrolj-tic. 
^o  BatteUi  and  Stern,  Biochem.  Zeit.,  1910  (29),"  130. 


OXIDIZING  ENZYMES  67 

used  up.  Its  rariRo  of  actiou  is  limited,  for  Jacoby  found  it  mthout  effect  upon 
acetic  acid  and  stearic  acid. 

Tyrosinase. — Tliis  cnzyine,  which  is  found  both  in  animal  and  plant  tissues, 
is  particularly  iiitcrostinu;  in  relation  to  the  formation  of  pigments.  Bcrtrand 
found  tliat  the  traiisforiuatiou  of  the  juice  of  lac-yicldin^!;  plants  into  the  black 
lacquer  was  liroufiht  al)()ul  by  the  action  of  an  oxi<lizinK  ferment,  Ificrane,  upon  an 
easily  oxidized  sulistance,  laccnl,  which  is  a  menil)er  of  the  aromatic  series.  He 
later  found  in  a  numl)er  of  j)lants  an  enzyme  acting  on  tyrosine,  distinct  from  the 
laccase,  whicii  he  named  tyrosinase,  liiedcrman  later  found  tyrosinase  in  the 
intestinal  fluid  of  meal  worms,  v.  Fiirth  and  Schneider  found  a  similar  enzyme 
in  the  hemolymph  of  insects  and  arthropods,  which  explains  its  darkening  when 
exposed  to  air.  This  enzyme,  as  obtained  from  different  sources,  is  not  always 
specific  for  tyrosine,  frequently  oxidizing  other  substances.  As  yet  the  chemical 
processes  and  end  results  of  the  oxidation  of  tyrosine  by  tyrosinase  are  unknown. 
Bach'-"  obtained  evidence  that  tyrosinase  is  not  a  specific  oxidizing  enzyme,  but 
consists  of  an  aminoacidase,  which  disintegrates  the  tyrosine  and  makes  it  sus- 
ceptible to  the  action  of  phenolase  which  is  the  oxidizing  agent,  v.  Fiirth  and 
Schneider  found  the  product  of  oxidation  of  tyrosine  by  animal  tyrosinase  related 
to  certain  of  the  melanins  of  animal  tissues,  and  believe  that  tyrosinase  is  respon- 
sible for  the  production  of  many  normal  pigments.-**  In  the  ink-sacs  of  the  squid, 
which  eject  an  inky  fluid  containing  melanin-like  pigment,  tyrosinase  was  also 
found,  corroborating  tliis  hypothesis,  and  it  is  probable  that  tyrosinase  in  the  skins 
of  animals  is  responsible  for  their  pigmentation. -^  Bacteria  also  contain  tyrosi- 
nase,'" and  this  or  similar  enzymes  seem  to  be  present  in  melano-sarcomas.-'^ 

Gonnermann-'-  found  that  tyrosinase  from  beet-root  produced  homogentisic 
acid  by  acting  on  tyrosine,  wliich  is  of  interest  in  connection  with  the  congenital 
hereditary  disease,  alkaptonuria  (q.  v.),  in  which  the  urine  becomes  dark  upon 
exposure  because  of  the  presence  of  homogentisic  acid.  The  action  of  tyrosinase 
upon  the  aromatic  radicals  of  proteins  is  of  great  importance  in  the  study  of  both 
physiological  and  pathological  pigment  formation,  and  hence  has  received  ex- 
tensive study,  which  ^^•ill  be  found  fully  described  in  the  monograph  by  Kastle 
(loc.  cit.)^'  and  under  the  appropriate  subjects  in  subsequent  chapters. 

Other  Oxidizing  Enzymes. — Of  the  great  number  of  other  less  studied  oxidizing 
enzymes  little  can  be  definitely  stated.  Some  consider  that  they  are  largely  dif- 
ferent manifestations  of  the  action  of  one  oxidizing  ferment,  but  against  this  view 
Jacoby  mentions  that  they  occur  distributed  imequally  in  different  organs,  can  be 
separated  from  each  other,  and  they  cause  different  reactions.  For  the  catalase 
and  for  laccase  (which  produces  the  Japanese  lacquer  by  an  oxidizing  process) 
and  perhaps  for  other  oxidizing  ferments,  iron  and  manganese  may  be  essential 
constituents.  Of  particular  significance  for  pathology  are  the  enzymes  which 
accomplish  the  oxidation  of  purines  to  uric  acid  and  the  subsequent  destruction  of 
uric  acid.  These  are  discussed  in  Chapter  xxiii.  Also  the  enzymatic  oxidation 
and  reduction  of  /3-oxybutyric  acid  and  aceto-acetic  acid  in  the  liver,  as  studied  by 
Dakin  and  Wakeman,'-^  are  of  great  importance  in  acidosis  {q.  v.). 

Reducing  enzymes  have  not  yet  been  satisfactorily  demonstrated.^^  It  is 
possible  that  they  do  not  exist,  and  that  the  intracellular  reductions  that  are 
carried  on  within  the  cells  are  brought  about  by  simple  chemical  reactions  inde- 
pendent of  catalysis.  The  best  known  intracellular  reduction  is  that  of  methylene 
blue,  ^5  which  can  be  readily  studied  experimentally  because  the  blue  color  dis- 

"Biochem.   Zeit.,   1914   (60),  221. 

=8  Bloch  (Zeit.  physiol.  Chem.,  1917  (100),  226)  describes  under  the  name 
dopaoxidase  an  enzj-me  present  in  the  protoplasm  of  the  basal  epidermal  and  hair 
follicle  cells,  which  acts  specifically  on  3,  4 — dihydroxyphenylalanine,  causing 
oxidation  and  condensation  with  formation  of  a  dark  brown  or  black  pigment. 

29  Meirowsky,  Cent.  f.  Path.,  1909  (20),  301. 

30  Lehmann  and  Sano,  Arch.  f.  Hyg.,  1908  (67\  99. 

31  Alsberg,  Joiu-.  Med.  Res.,  1907  (16),  117;  Neuberg,  Virchow^'s  Archiv.,  1908 
(192),  514;  Gessard,  Compt.  Rend.  Soc.  Biol.,  1902  (54),  1305. 

"Pfluger's  Arch.,   1900  (82),  289. 

"Jour.  Amer.  Med.  Assoc,  1910  (54),  1441. 

"  See  Heffter,  Arch.  exp.  Path.  u.  Pharm.,  1908,  Suppl.,  p.  253. 

36  See  Thunberg,  Skand.  Arch.  Physiol.,  1917  (35),  163. 


68  ENZYMES 

appears  on  reduction  of  tlie  dye.  It  is  open  to  question  if  this  particular  reduction 
is  due  to  a  reducing  enzyme.  According  to  Ricketts^*  the  reduction  depends  upon 
two  bodies,  one  therniostabile,  the  other  therrnolabile,  recalling  the  reaction  of 
complement  and  amboceptor.  Strassner"  found  evidence  that  the  .SH  groups  of 
the  tissues  are  responsible  for  the  reduction  of  methylene  blue;  their  activity 
is  impaired  by  heating,  but  a  thermostable  element  of  tissues  augments  the  re- 
ducing acti\ity  of  SH  compounds,  thus  corroborating  and  explaining  the  observa- 
tions of  Ricketts.  Harris,'*  however,  believes  that  the  evidence  for  the  existence 
of  a  true  reducing  enzyme  is  as  good  as  for  most  other  cellular  enzj-mes.  An  en- 
zyme has  been  found  in  the  liver,  muscle  and  kidney  which  transforms  aceto-acetic 
acid  into  l-/3-oxybutyric  acid,  and  called  ketoreductase  (Friedmann  and  Maase)." 

Oxidizing  Enzymes  in  Pathological  Processes. — Although  the 
oxidizing  enzymes  undoubtedly  play  an  important  part  in  pathological 
conditions,  they  have  been  but  little  investigated  from  this  stand- 
point. Jacoby  found  that  they  did  not  disappear  from  the  degen- 
erated liver  in  phosphorus  poisoning  or  in  diabetes,  or  «-hen  the  liver 
undergoes  self-digestion,  which  speaks  against  Spitzer's  contention  that 
oxidase  is  a  nucleoprotein.^°  Schlesinger^^  found  that  it  is  less  in 
amount  in  livers  of  children  dead  from  gastro-intestinal  diseases  than 
in  normal  livers,  as  also  did  Brtining."*^  I  am  inclined  to  believe  that 
fatty  metamorphosis,  when  brought  about  by  poisons,  is  often  due 
to  inhibition  of  the  oxidizing  enzymes  (v.  fatty  metamorphosis), 
although  I  found  that  livers  the  seat  of  the  most  profound  fatty  de- 
generation showed  no  evident  impaiiment  of  their  power  to  oxidize 
xanthine  and  uric  acid.'^^  Buxton'*^  failed  to  find  in  tumors  any  en- 
zyme giving  the  guaiac  test  alone,  but  found  enzymes  that  did  so  in 
the  presence  of  H2O2  (peroxidases).  Catalase  was  present,  but  no 
very  positive  reactions  for  oxidizing  enzymes  w^ere  obtained  by  the 
indo-phenol  reaction,  the  hydrochinon  reaction,  or  with  tjTosine  for 
tyrosinase,  v.  Fiirth  and  Jerusalem''^  have  found  evidence  that  the 
melanin  of  melanotic  tumors  of  horses  is  produced  by  tyrosinase. 
Peroxidase  has  been  demonstrated  in  the  granules  of  pus  cells  (Fisehel) .  *^ 

Meyer ^^  found  that  leucocytes,  whether  from  pus  or  leukemic  or 
pneumonic  blood,  contained  a  substance  oxidizing  guaiac  directly, 
without  the  presence  of  H2O2,  which  is  not  liberated  until  the  cells 
are  destroyed.  By  microchemical  reactions  oxidases  have  been  found 
present  in  the  myelocytes  and  nucleated  erythrocytes  in  leukemia,  be- 

'^  Jour,  of  Infectious  Diseases,  1904  (1),  590. 

"  Biochem.  Zeit.,  1910  (29),  295. 

=8  Biochem.  Jour.,  1910  (5),  143. 

="»  Biochem.  Zeit.,  1912  (27),  474;  1913  (55),  458. 

*"  Duccheschi  and  Almagia  (Arch.  ital.  Biol.,  1903  (39),  29)  also  found  the 
aldehydase  in  livers  of  phosphorus  poisoning  usually  no  less  abundant  than  in 
normal  livers. 

*'  Hofmcister's  Beitr.,  1903  (4),  87. 

*-^  Monat.  f.  Kinderheilk.,  1903  (2),  129. 

"Jour.  Exper.  Med.,  1910  (12),  607 

**  Jour.  Med.  Research,  1903  (9),  350. 

^6  Hofmeister's  Beitr.,  1907  (10),  131. 

"Wien.  klin.  Woch.,  1910  (23).  15.57. 

<'  Munch,  med.  Woch.,  1903  ('A)),  14S9. 


ENDOrilEXOL  REACTION  ()9 

ing  al)S(Mit  from  the  polynuclcar  colls.  '^  The  observation  of  Natalie 
Siebci-*''  that  oxidases  of  the  blood  and  of  vegetable  origin  destroy 
diphtheria  toxin  rapidly,  and  also  tetanus  toxin  and  ricin,  has  been 
confirmed  by  Loewenstein  as  far  as  destruction  by  peroxide,  with  or 
without  the  presence  of  catalase,  is  concerned.  Oxidation  is  un- 
doubtedly an  important  process  in  defending  the  body  against  other 
forms  of  poisons,  including  the  so-called  "fatigue  toxins,"  and  Battelli 
and  Stern  consider  that  all  the  oxidizing  enzymes  so  far  definitely 
identified  are  concerned  only  in  protective  processes.  Schmidt^°  has 
found  that  liver  extracts  render  certain  morphin  derivatives  non- 
poisonous  by  oxidation.  Oxalic  acid  and  poisonous  fatty  acids  are 
also  oxidized  into  harmless  substances;  phosphorus  and  sulphur  are 
oxidized  into  their  acids,  which  are  then  neutralized.  Indole  and 
skatole  are  oxidized  into  less  harmful  substances. 

The  Indophenol  Reaction. 5' — Alplia-naphthol  and  dimothyl-para-pheiiylcndia- 
min,  when  brought  together  in  alkaline  solution,  become  oxidized  in  the  presence 
of  air  and  form  an  insoluble  blue  dye,  indophenol.  This  reaction  is  greatly 
accelerated  by  oxidizing  agents,  and  it  has  been  found  that  certain  tissues  pos- 
sess this  property,  hence  the  indophenol  synthesis  has  been  used  for  microchemical 
study  of  the  presence  and  distribution  of  oxidizing  enzymes  in  cells.  As  the  in- 
tracellular agent  which  causes  this  reaction  is,  however,  so  resistant  to  heat  and 
chemicals  that  it  can  be  demonstrated  in  sections  fixed  in  formalin  and  prepared 
by  the  ordinary  paraffin  imbedding  method  (Dunn),  there  is  room  for  much  doubt 
as  to  whether  it  represents  a  true  enzyme,  although  it  has  been  considered  identical 
with  phenolase.^-  In  the  presence  of  small  amounts  of  peroxide  the  granules  of 
leucocytes  and  myelocytes  are  stained  with  alphanaphthol  alone,  which  Graham** 
interprets  as  oxidation  by  an  enzyme  of  the  peroxidase  type.  By  using  a  rf-naph- 
thol  and  paraphenylenediamine  and  staining  for  long  periods,  Menten*^  has 
obtained  positive  reactions  in  all  tissues,  and  has  observed  a  similar  effect  with 
cholesterol  esters.  The  evidence  obtained  indicates  that  the  oxidation  is  not 
determined  by  enzymes  but  apparently  is  dependent  on  adsorption  phenomena 
taking  place  on  intracellular  surfaces. 

The  indophenol  reaction  is  observed  best  in  the  granules  of  neutrophile  leu- 
cocytes of  blood  and  in  myeloid  cells  of  bone  marrow,  leukemic  blood  and  fetal 
organs;  eosinophiles  and  basophile  leucocytes  also  give  reactions,  but  not  Ij'mpho- 
cytes,  platelets,  megakarocytes,  plasma  cells,  mature  orj-throcytes,  or  most  fixt 
tissue  cells."  By  using  alkali-free,  unfixt  tissues  Gierke  found  granules  present 
in  tissue  cells  generally,  and  Griiff  states  that  they  occur  in  proportion  to  the 
metabolic  activity  of  the  cells;  they  are  abundant  in  carcinomas,  scanty  in  sarcoma 
and  connective  tissue  growths  generally,  are  not  destroyed  in  cloudy  swelling  or 
fatty  changes,  but  disappear  in  infarcts  and  autolyzing  tissues,  and  in  tissues 
asphyxiated  with  illuminating  gas.*^  Lung  tissue  is  especially  poor  in  this  form 
of  oxidative  activity,^'  but  giant  cells  of  tubercles  contain  oxidase  granules.*^ 

■'^  Fiessinger  and  Roudowska,  Arch,  de  med.  exper.,  1912  (24),  585. 

"  Zeit.  physiol.  Chem.,  1901  (32),  573. 

^°  Dissertation,  Heidelberg,  1901. 

^1  Literature  given  bv  Schultze,  Ziegler's  Beitr.,  1909  (45),  127;  Dunn,  Jour. 
Path,  and  Bact.,  1910  (15),  20;  Graff.  Frankfurter  Zeit.  f.  Path.,  1912  (12),  35S; 
Rosenthal,  Arch.  Int.  Med.,  1917  (20),  185. 

"  Bach  and  Maryanovitsch,  Biochem.  Zeit.,  1912  (42),  417. 

"Jour.  Med.  Res.,  1916  (35),  231. 

"Jour.  Med.  Res.,  1919  (40),  433. 

"See  Dunn,  Quart.  Jour.  Med.,  1913  (li),  293. 

56  See  Ivlopfer,  Zeit.  exp.  Pharm.,  1912  (11),  4t)7. 

"  Weiss,  Wien.  klin.  Woch.,  1912  (25),  097. 

='«Makino,  Verb.  Japan.  Path.  Gesell.,  1915  (5),  71. 


70  ENZYMES 

During  experimental  pneumococcus  septicemia  the  indophenol  oxidase  reaction 
is  decreased  in  the  tissues.** 

The  nature  of  the  granules  that  exhibit  the  stain  is  unknown,  but  as  indophenol 
blue  is  a  good  fat  stain  it  is  probable  that  the  stained  granules  are  lipoidal,  and  it 
may  well  be  that  they  are  not  the  site  of  the  oxidative  action,  but  merely  selectively 
stained  lipoids  in  cells  capable  of  forming  the  indophenol  blue.  These  so-called 
"oxidase"  granules  have  been  divided  into  stable  and  labile,  the  former  staining 
b}^  the  Winkler  oxidase  reaction,  the  latter  by  Gierke's  reaction.  The  granules 
seem  to  pass  from  the  leucocytes  into  their  environment.  When  animals  are 
exposed  to  x-raj's  the  stable  granules  are  destroyed  sooner  than  the  labile  granules. 
The  relation  of  the  granules  to  other  cell  granules  is  undetermined,  and  their  dis- 
tribution is  not  identical  with  the  granules  that  take  the  vital  stains.  Katsunuma*" 
considers  that  they  are  probably  not  permanent  specific  structures,  but  transitional 
alterations  produced  in  functional  activity  of  the  protoplasm. 

Glycolytic  Enzymes.^' — The  oxidation  of  sugar  bj'  the  tissues,  which  is  one  of 
the  chief  sources  of  energy  in  the  animal  body,  presumably  takes  place  through 
several  steps.  Of  these,  it  is  believed  by  some  that  the  first  is  the  formation  of 
glycuronic  acid — 

O  O 

II  II 

CH20H-(CHOH)4C-H  +  02  =  C00H-(CH0H)4C  -  H  +  HA 
(glucose)  (glycuronic  acid) 

but  the  subsequent  changes  which  involve  decomposition  of  the  straight  chain  are 
not  at  present  understood.  Attempts  to  isolate  from  various  organs  an  enzyme 
oxidizing  glucose,  particularly  from  the  pancreas,  muscle,  and  liver,  have  led  to 
varying  results  and  much  dissension,  but  it  is  probable,  because  of  these  failures, 
that  no  such  enzyme  exists  in  quantities  sufficient  to  account  for  the  amount  of 
sugar  combustion  that  is  normalh'  accomplished.  O.  Cohnheim^-  attempted  to 
explain  the  failures  by  his  observation  that  the  pancreas  produces  a  substance 
that  activates  an  inactive  glycolytic  enzyme  in  the  muscles,  liver,  and  probably 
in  other  organs.  This  work  is  not  generally  accepted,  so  we  are  still  in  the  dark 
as  to  how  the  carbohydrate  oxidations  are  accomplished.     (See  Chapter  xxiv.) 

Lipase  " 

Lipase  is  probably  present  in  greater  or  less  amount  in  all  cells. 
In  the  discussion  of  the  reversible  action  of  enzymes  (see  page  51) 
the  modern  conception  of  fat  metabolism  has  been  explained,  which 
considers  it  to  depend  upon  the  existence  of  lipase  in  the  cells  and  fluids 
throughout  the  body.  On  account  of  the  technical  difficulties  in  the 
way  of  using  higher  fats,  such  as  triolein,  in  experimental  work,  the 
esters  of  lower  fatty  acids  have  generally  been  used,  particularly  ethyl 
butyrate,  salicylic  acid  esters,  and  glycerol  triacetate.  Enzymes  split- 
ting ethyl  butyrate,  and  other  esters  {esterases) ,  have  been  demonstrated 
in  practically  all  tissues  examined,  the  names  of  Kastlc  and  Loeven- 
hart  in  this  country,  and  Hanriot  in  France,  being  particularly  con- 
nected with  this  work.     What  the  relation  of  these  esterases  may  be 

f-s  Medigreceanu,  Jour.  Exp.  Med.,  1914  (19),  303. 

«»  Verb.  Jap.  Path.  Ges.,  191G  ((>),  76. 

8' Also  discussed  under  "Diabetes,"  chap.  xxiv.  As  glycolysis  by  blood  and 
tissues  can  occur  witliout  oxygen,  Battclli  and  .Stern  exclude  tlie  glycolytic  from 
the  oxidizing  enzvmes. 

•^'^  Zeit.  physiol.  ('hem.,  1903  (39),  336;  also  see  Simpson,  Hiochcin.  .lour.,  1910, 
(5),  126. 

"'  For  literature  ou  lii)ase  see  Connstein,  Ergcbnis.se  Physiol.,  190  I  (3,  Abt.  1), 
194;  concerning  the  l)ehavi()r  of  lipase  sec  Tavlor,  Jour.  BioL  ('hem.,  1906  (2), 
103;  Palk,  Proc.  Natl.  Acad.,  1915  (1),  136;  Science,  1918  (47),  423. 


LIPASE  71 

to  the  enzyme  splitting  fats,  the  triu;  lipase,  is  not  yet  known.  Much 
of  the  work  so  far  reported  on  the  occurrence  of  lipase  in  tissues  is  of 
questionable  value,  especially  as  to  quantitative  results,  because  of 
faulty  methods.  SaxP*  points  out  and  avoids  some  of  these  errors, 
and  finds  that  during  autolysis  of  tissues  the  splitting  of  the  natural 
fats  present  in  the  cells  is  but  slight;  simple  esters  are  attacked  more, 
especially  amyl-salicylate ;  muscle  and  blood  are  the  least  active 
tissues.  Most  authors  agree  that  lymphoid  cells  are  especially  rich  in 
lipolytic  enzymes. ^^  In  the  serum  of  normal  individuals  the  esterase 
content  seems  to  be  quite  constant,^''  and  Quinan"  found  the  tissue 
content  also  constant,  the  liver  containing  about  twice  as  much  as 
ttie  kidnej'  and  over  three  times  as  much  as  the  muscle.  He  states 
that  different  parts  of  the  brain  have  characteristic  lipase  activity 
(butyrase).*^^  Thiele^^  has  found  that  blood,  chyle,  and  various 
tissues  also  contain  an  enzyme  which  can  hydrolyze  lecithin,  but  except 
in  the  pancreas  i  does  not  hydrolyze  neutral  fats.  The  brain  contains 
enzymes  hydrolyzing  mono-  and  triacetin,  lecithin  and  cephalin.'"' 

Little  is  known  about  the  part  played  by  lipase  in  pathological  con- 
ditions. According  to  Achard  and  Clerc,^'  the  amount  of  spUtting  of 
ethyl  butyrate  by  the  blood-serum  is  lessened  in  most  diseases,  and  in- 
creases and  decreases  with  the  health  of  the  patient;  accorchng  to 
Pribram^2  a^id  SagaP^  it  is  increased  in  the  blood  during  fevers. 
Clerc^^  found  that  acute  arsenic,  phosphorus  and  diphtheria-toxin 
poisoning  increased  this  property  of  the  serum,  while  chronic  poison- 
ing and  staphylococcus  intoxication  lowered  it.  Somewhat  similar 
results  were  obtained  by  Grossmann,''*  but  Saxl  found  no  increased 
activity  in  phosphorus  poisoning.  Using  the  ethyl  butyrate  test, 
Winternitz  and  Meloy^^  found  that  the  more  nearly  normal  an  organ 
is  the  more  cleavage  of  the  ester;  lipolytic  activity  is  low  at  birth, 
increases  rapidly  during  the  first  few  days  of  life,  and  does  not  de- 
crease in  old  age.  There  is  a  decline  in  activity  of  tissues  in  diabetes, 
tuberculosis,  and  the  toxemia  of  pregnancy,  in  the  livers  of  passive 
congestion  and  fatty  degeneration,  in  the  pneumonic  lung  and  the 
cirrhotic  liver.     After  taking  food  there  is  a  slight  increase  in  esterase, 

e^Biochem.   Zeit.,   1908  (12),  343. 

^^  The  distribution  of  lipases  in  different  species  of.'animals  and  their^various 
organs  has  been  investigated  by  Porter,  Miinch.  med.  Woch.,  1914  (61),  1774. 

«  Sagal,  .Jour.  Med.  Res.,  1916  (34),  231. 

«^  Ibid.,  1915  (32),  45. 

"  Ibid.,  1916  (35),  79. 

«^  Biochem.  Jour.,  1913  (7),  275. 

"  English  and  MacArthur  (.Jour.  Amer.  Chem.  Soc,  1915  (37),  653),  who 
have  also  found  in  sheep  brain,  erepsin,  amylase,  catalase,  enzymes  decomposing 
arbutin  and  salol,  probably  pepsin  and  trypsin,  but  not  peroxidase,  oxidase, 
reductase,  guanase,  urease  or  rennin. 

"  Compt.  Rend.  Soc.  Biol.,  1902  (54),  1144. 

"  Cent.  inn.  Med.,  1908  (29),  81. 

"Compt.  Rend.  Soc.  Biol.,  1901  (53),  1131. 

'*  Biochem.  Zeit.,  1912  (41),  181. 

'Uour.  Med.  Res.,  1910  (22),  107. 


7.2  ENZYMES 

reaching  a  maximum  in  three  hours. '^^  Whipple''^  finds  the  blood 
Hpase  (butyrase)  increased  whenever  there  is  injury  to  the  liver,  such 
as  in  chloroform  anesthesia  and  puerperal  eclampsia;  it  is  lowered 
in  cirrhosis.  Poulain^^  found  that  the  butyric-splitting  power  of 
lymph-glands  draining  infected  areas  was  decreased.  Fischcr^^ 
observed,  in  a  case  of  extreme  lipemia  in  diabetes,  that  the  lipolytic 
power  of  the  blood  was  absent.  The  lipase  of  lipomas  presents  no 
demonstrable  difference  from  that  of  ordinary  fatty  areolar  tissues.*" 

Lipase  has  also  been  demonstrated  in  pus  by  a  number  of  ob- 
servers,*^ who  agree  that  there  is  more  in  exudates  than  in  transu- 
dates. Zeri*2  found  lipase  in  the  urine  only  when  pus  or  blood  was 
also  present,  but  Pribram  and  Loewy*^  found  it  in  nephritis,  con- 
gestion, polyuria  and  other  conditions.  Lorenzini,**  however,  re- 
ports that  in  albuminuria  the  lipase  content  of  the  urine  is  reduced, 
in  common  with  other  enzymes,  there  being  a  simultaneous  accumula- 
tion of  enzymes  in  the  blood. 

Fiessinger  and  Marie*^  contend  that  the  lymphocytes  of  exudates 
are  the  chief  source  of  lipase,  and  suggest  that  this  may  be  of  effect 
in  defense  against  the  fatty  tubercle  bacilli.  Toxins  were  found  by 
Pesci*®  to  increase  the  butyrase  but  not  the  other  lipases  of  liver 
tissue.  In  syphilis  the  lipolytic  activity  of  the  serum  is  increased,*' 
which  may  be  related  to  Bergell's**  observation  on  the  origin  of  lipase 
in  lymphocytes  (corroborating  Fiessinger  and  Marie).  Jobling  and 
Bull**  state  that  a  specific  serum  lipase  increase  occurs  in  animals 
immunized  to  red  corpuscles,  and  that  this  lipase  has  to  do  with 
hemolysis;  but  MendeP**  found  no  evidence  that  hemolj'sis  by  ricin  is 
related  to  lipase.  Abderhalden  and  Rona*^  found  that  excess  feeding 
of  fats  leads  to  an  increase  in  the  lipase  of  the  blood. 

The  part  played  by  lipase  in  fatty  degeneration  must  be  of  great 
importance,  but  as  yet  it  has  been  little  considered,  except  that  Loeven- 
hart,  and  Duccheschi  and  Almagia*^  found  no  appreciable  difference 

'"  Jobling  et  al,  Jour.  Exp.  Med.,  1915  (22),  129. 

"  Whipple  et  al,  Bull.  Johns  Hopkins  Hosp.,  1913  (2-4),  207  and  357. 

'«  Comp.  Rend.  Soc.  Biol.,  1901  (53),  786. 

'^Virchow's  Arch.,   1903  (172),  218. 

'"  Wells,  Arch.  Int.  Med.,  1912  (10),  297. 

"  Achalme,  Comp.  Rend.  Soc.  Biol.,  1899  (51),  5GS;  Zeri,  II  Policlinico,  1903 
(10),  433;  Memmi,  Clin.  Med.  Ital.,  1905  (44),  129. 

82  II  PoUclinico,  1905  (12),  733. 

8'  Zeit.  phvsiol.  Chem.,  1912  (76),  489. 

"'Policlinico,  1915  (22),  358. 

"  Compt.  Rend.  Soc.  Biol.,  1909  (68),  177.  See  also  Resell,  Dout.  Arrh.  klin. 
Med.,  1915  (118),  179. 

8«  PatholoM;ica,  1912  (3),  207. 

8' Citron  and  Reicher,  B(>rl.  klin.  Woch.,  1908  (45),  1398. 

88  Miinch.  nicd.  Wocli.,  1909  (56),  64. 

89  Jour.  Kxp.  Med..  l',M2  (16),  483. 
«<•  Arch.  Fisiol.,  1<)09  (7),  1()S. 
"Zeit.  plivsiol.  Chem.,  1911   (75),  30. 
"2  Arch.  Ital.  Biol.,  1903  (39),  29. 


AMYLASE  OR  DIASTASE  73 

in  the  lipase  eontoiit  of  normal  and  pliosphoius-poisoned  livers,  but 
in  chloroform  poisoning  Quinan'''  found  a  decrease  in  the  butyrase 
of  the  liver,  although  it  was  increased  in  the  kidneys  and  muscles. 
This  question  will  be  considered  more  fully  in  discussing  fatty  meta- 
morphosis. 

An  improved  method  of  testing  for  lipase  action  has  been  devised 
by  Rona  and  ]\Iichaclis,  by  measuring  the  change  in  surface  tension 
caused  by  hydrolysis  of  a  soluble  ester,  usually  tributyrin.  Using  this, 
Bauer'*  found  that  every  human  serum  contains  fat-splitting  enzymes, 
which  are  greatly  decreased  in  carcinoma  and  advanced  phthisis,  some- 
what decreased  in  syphilis  and  exophthalmic  goitre,  and  increased  in 
early  pulmonary  tuberculosis.  Caro'^  found  a  decrease  in  all  cases 
of  cachexia,  but  there  was  no  relation  between  the  lipolytic  enzyme 
and  the  blood  picture.  The  blood  contains  no  thermostable  antilipase 
analogous  to  the  antitrypsin.  Red  corpuscles  are  said  to  contain  an 
enzyme  sphtting  cholesterol  esters,  "cholesterase."^^  In  leucocytes  a 
"lipoidase"  has  been  found  by  Fiessinger  and  Clogne"  that  splits 
choline  out  of  lecithin. 

Fat  necrosis,  resulting  from  the  escape  of  pancreatic  juice  into  the 
peripancreatic  tissues  and  abdominal  cavitj^  undoubtedly  is  largely 
the  result  of  lipase  action.  (See  "Fat  Necrosis,"  Chapter  xv,  for 
complete  consideration.) 

Amylase  or  Diastase"" 

Although  under  ordinary  conditions  starch  is  not  supposed  to  enter 
the  blood  stream  and  tissues,  yet  all  tissues  and  body  fluids  are  capable 
of  hydrolyzing  starch.  Apparently  the  amylase  is  derived  from  the 
pancreas  and  salivary  glands,  and  possibly  from  many  or  all  other 
tissues  (King),  but  it  is  not  quantitatively  related  to  the  amount  of 
carbohydrate  in  the  diet  of  a  species  or  an  individual  (Carlson  and 
Luckhardt).  In  the  blood  it  occurs  in  the  albumin  fraction.'*  There 
is  disagreement  in  the  literature  as  to  the  variations  in  amount  of 
amylase  in  the  blood  during  disease,  and  little  information  concerning 
its  distribution  in  the  tissues.  Normally  the  kidneys  and  Uver  seem 
to  be  most  active  and  Winslow  says  that  all  glycogen-containing 
organs  produce  diastase.  During  acute  infections  the  blood  amylase  is 
increased,  presumably  coming  from  the  leucocytes  (King).  It  is 
greatly  increased  when  the  pancreas  is  acutely  inflamed  or  injured 

93  Jour.  Med.  Res.,  1915  (32),  73. 

sMVien.  klin.  Woch.,  1912  (25),  1376  (bibliography). 

"  Zeit.  klin.  Med.,  1913  (7S),  286. 

3«See  Cvtronberg,  Biochem.  Zeit.,  1912  (45),  281. 

9^  Compt.  Rend.  Acad.  Sci.,  1917  (165),  730. 

""  Literature  given  by  Watanabe,  Anier.  Jour.  Physiol.,  1917  (45),  30;  Geyelin, 
Arch.  Int.  Med.,  1914  (13),  96;  Stocks,  Quart.  Jour.  Med.,  1916  (9),  216;  McClure 
and  Pratt,  Arch.  Int.  Med.,  1917  (19),  568;  Winslow,  Hospitalstidende,  1918  (61) 
832 

""sSatta,  Arch.  Sci.  Mt'd.,  1915  (.39),  46. 


74  ENZYMES 

(Stocks).  In  diabetes  it  is  ordinarily  increased,  but  not  in  syphilitic 
diabetes. ^^  Intravenous  or  subcutaneous  injection  of  starch  is  said 
to  increase  the  blood  amylase,  presumably  as  a  defensive  reaction 
(Abderhalden),  but  the  amylase  ordinarily  in  the  blood  seems  to  be  a 
waste  substance  on  its  way  to  excretion,  rather  than  a  functionating 
enzyme  of  the  blood.  There  appears  to  be  no  normal  antiamylase  in 
the  blood.  Starch  granules  taken  up  by  phagocytes  show  a  glycogen 
reaction  after  some  time,  suggesting  that  these  cells  have  intracellular 
diastases.^ 

Because  of  possible  diagnostic  significance,  the  amylolytic  activity 
of  the  urine  has  been  particularly  studied,  and  found  normally  to  be 
approximately  constant  for  24  hour  specimens  of  the  same  individual. - 
Anything  impairing  the  excretory  capacity  of  the  kidney  decreases  the 
urinary  amylase,  although  sometimes  when  the  urine  contains  blood, 
pus,  or  much  albumen  there  may  be  an  increased  amylase  excretion  in 
spite  of  diminished  functional  activity.  There  may  be  an  increase  in 
the  amylase  in  the  blood  when  the  urinary  amylase  is  decreased,  but 
with  normal  kidneys  increase  of  the  blood  amylase  causes  an  increase 
in  the  urine;  hence,  acute  pancreatic  diseases  cause  an  increased 
urinary  amylase  (Stocks),  but  this  is  not  constant  (McClure  and 
Pratt).  In  diabetic  urine  it  is  said  to  be  usually  decreased,  but  this 
is  mostly  accounted  for  by  the  dilution  of  the  urine.  Parenteral  in- 
jection of  starch  causes  a  marked  increase  in  the  amount  of  diastase  in 
the  urine  (King).^ 

99  De  Niord  and  Schreiner,  Arch.  Int.  Med.,  1919  (23),  484. 

>  Okazaki,  Sei-I-Kwai  Med.  Jour.,  1917  (36),  101. 

^  In  infants  the  urine  amylase  is  low  (McClure  and  Chancellor,  Zeit.  Kinder- 
heilk.,  1914  (11),  483.  Fetal  blood  contains  much  less  than  the  maternal  blood 
(Kito,  Amer.  Jour.  Physiol.,  1919  (48),  481). 

3  Proc.  Soc.^Exp.  Biol.,  1917  (15),  101. 


CHAPTER  III 

ENYZMES  (Continued) 

INTRACELLULAR  PROTEASES'  (PROTEOLYTIC  ENZYMES),  INCLUDING 
A  CONSIDERATION  OF  AUTOLYSIS 

To  what  extent  synthesis  of  proteins  goes  on  in  the  body  is  still  a 
problem;  still  more  uncertain  is  the  part  played  by  reversible  action 
of  proteases.  There  is  evidence  enough  that  somewhere  in  the  body 
the  amino-acids  can  be  rebuilt  into  protein,  for  several  investigators 
have  succeeded  in  keeping  animals  in  nitrogenous  equilibrium  by  feed- 
ing them  products  of  proteolysis  that  contained  no  proteins  whatever, 
and  as  the  proteins  of  the  animal  body  are  being  broken  down  in- 
cessantly, it  must  be  that  they  were  replaced  by  synthesis  of  the  non- 
protein material  fed  to  the  animals.  In  addition,  it  has  long  been 
questioned  whether  amino-acids  absorbed  from  the  intestines  are  not 
resynthesized  into  proteins  while  passing  through  the  intestinal  wall. 
Cohnheim  found  that  in  the  intestinal  epithelium  there  is  an  enzyme, 
erepsin,  capable  of  splitting  albumoses  and  peptones  into  the  amino- 
acids,  which  enzyme  presumably  exists  for  the  purpose  of  securing 
complete  cleavage  of  all  ingested  proteins  into  their  ultimate  "build- 
ing stones."  This  may  be  looked  upon  as  a  provision  to  reduce  all 
varieties  of  proteins  to  their  common  elements,  so  that  the  body  by 
quantitative  selection  can  resynthesize  them  into  its  own  types  of 
protein,  for,  as  is  well  known,  foreign  proteins  {e.  g.  egg-albumin) 
introduced  directly  into  the  blood  stream  cannot  be  utilized,  but  are 
excreted  unaltered  in  the  urine. ^  As  was  shown  for  lipase,  the  as- 
sumption that  such  synthesis  occurs  as  a  normal  physiological  process 
by  reverse  enzyme  action,  requires  that  the  proper  enzymes  be  present 
in  the  cells  throughout  the  body,  and  within  recent  years  it  has  been 
abundantly  demonstrated  that  such  is  the  case. 

For  over  half  a  century  it  has  been  known  that  amebse  digest  solid 
proteins  within  their  bodies,  but  it  is  only  within  a  few  years  that 
proteolytic  enzymes  have  been  definitely  isolated  from  them.  It  has 
been  much  the  same  with  the  intracellular  proteases  of  the  higher 
organisms.     In    1871    Hoppe-Seyler  referred   to  the  liquefaction  of 

^  As  the  possibility  exists  that  ferments  which  digest  proteins  may  be  able  to 
perform  a  certain  amount  of  synthesis  of  proteins,  the  term  "proteolytic  enzyme" 
seems  to  be  less  suitable  than  the  term  "'protease,"  which  merely  means  an  enzyme 
acting  on  proteins,  and  does  not  compel  us  to  accept  any  particular  view  as  to 
what  the  action  is. 

^  According  to  Austin  and  Eisenbrey  (Arch  Int.  Med.,  1912  (10),  305),  dogs 
on  a  nitrogen-free  diet  can  utilize  horse  serum  injected  intravenously. 

75 


76  ENZYMES 

dead  tissues  within  the  body  which  occurred  without  putrefaction, 
and,  as  he  noted,  resembled  the  effects  of  the  digestive  ferments.  It 
was  nearly  twenty  years  later  that  Salkowski^  showed  definitely  that 
this  softening  of  dead  tissues  was  really  brought  about  through  a  true 
digestion  by  intracellular  enzymes,  which  produced  the  same  splitting 
products  that  were  at  that  time  considered  characteristic  for  tryptic 
digestion  (leucine  and  tyrosine).  The  process  he  named  "autodiges- 
tion."  This  important  observation  remained  almost  unnoticed  for 
ten  years  more,  when  Jacoby,^  in  1900,  took  up  the  investigation  of 
this  matter  of  cellular  self-digestion,  and  after  this  the  importance  of 
the  principles  involved  became  for  the  first  time  generally  appreciated. 
Jacoby  rechristened  the  process  "autolysis,"  by  which  name  it  is  now 
commonly  known. 

AUTOLYSIS^ 

Autolysis  is  generally  studied  by  the  method  used  bj'  Salkowski, 
which  depends  upon  the  difference  in  the  susceptibility  of  bacteria 
and  of  enzymes  to  antiseptics.  The  organs  are  ground  to  a  pulp, 
placed  in  flasks  with  or  without  the  addition  of  water  or  dilute  acids, 
and  bacterial  action  is  prevented  by  the  addition  of  antiseptics  that 
are  not  poisonous  to  enzymes — 'toluene  and  chloroform  are  most  com- 
monly used.  It  is  possible  also  to  secure  organs  in  an  aseptic  con- 
dition and  to  permit  them  to  undergo  autolysis  without  the  use  of 
antiseptics,  but  the  practical  difficulties  are  such  that  this  method 
is  seldom  used — it  is  sometimes  designated  as  "aseptic  autolysis,  in 
contradistinction  to  antiseptic  autolysis  by  the  Salkowski  method.  In 
a  short  time  it  can  be  seen  that  digestive  changes  have  taken  place, 
particularly  if  comparisons  are  made  with  control  flasks  in  which 
the  enzymes  have  been  destroyed  by  boiling.  To  determine  the  rate 
of  autolysis  the  amount  of  nitrogen  that  remains  in  the  form  of  coagu- 
lable  compounds,  and  that  which  is  converted  into  soluble,  non- 
coagulable  compounds  (albumoses,  peptones,  ammonia  compounds, 
amino-acids,  etc.),  is  compared.  The  method  may  be  illustrated  by  a 
concrete  example:  A  given  specimen  of  emulsionized  liver  tissue  was 
permitted  to  digest  itself  for  twenty-two  days.  At  the  end  of  that 
time  39.4  per  cent,  of  the  nitrogen  was  still  contained  in  the  com- 
pounds that  remained  insoluble  or  became  so  after  the  autolysis  was 
stopped  by  boihng;  while  60.6  per  cent,  of  the  nitrogen  was  in  a 
soluble  form.  A  control  specimen  from  the  same  liver  was  boiled 
while  fresh  to  kill  the  enzymes,  and  then  let  stand  under  the  same 

3  Zeit.  f.  klin.  Med.,  1890,  supplement  to  Bd.  17,  p.  77. 

•"  Zeit.  f.  physiol.  Cheni.,  1900  (30),  149. 

5  Resviiiu';  of  literature  l)v  Salkow.ski,  Deut.schc  Klinik,  1903  (11),  147;  also  see 
Schlesinger,  HofnieistxT's  lieitriiKe,  1903  (4),  87;  Oswald.  Biochem.  Centr.,  1905 
(3),  365;  Levene,  Jour.  Ainer.  Med.  Assoc.,  1906  (46).  77t);  Nicolle,  Ann.  Inst. 
Pasteur.  1913  (27),  97;  von  Fiirth,  " Chemistry  of  Metal)olisiii,"  Ainer.  Transl., 
1916. 


PRINCIPLES  OF  AUTOLYSIS  77 

conditions.  In  this  specimen  90.4  per  cent,  of  tlie  nitrogen  was  in 
an  insoluble  form,  and  9.6  per  cent,  was  soluble.  Therefore,  over 
half  of  all  the  protein  of  the  liver  had  been  changed  into  non- 
coagulable  substances  in  the  course  of  about  three  weeks  (at  37°  C). 
Complete  disintegration  of  the  proteins  with  liberation  of  all  the 
amino-acid  complexes  is  probably  never  reached.  Of  45.8  grams  of 
amino-acids  present  in  100  grams  of  liver,  in  ten  days'  autolysis  there 
had  been  set  free  but  1.85  gm.,  after  30  daj^s  10.1  gm.,  and  after  50 
days  but  29.1  gm.  (Abderhaldcn  and  Prym.'')  Bj'  determining  the 
freezing  point  and  conductivitj''  of  autolyzing  mixtures,  valuable 
evidence  can  be  obtained  as  to  the  rate  of  change,  which,  in  some  cases, 
is  much  more  significant  than  the  usual  estimation  of  soluble  and  in- 
soluble nitrogen  (Benson  and  Wells^).  Titration  of  the  free  amino- 
acids  b}'  the  formaldehyde  method,  together  with  the  estimation  of 
proteose  and  peptone  nitrogen,  also  furnish  valuable  information, 
while  the  Van  Slyke  method  of  determining  free  amino-acids  is 
especially  useful  for  this  purpose. 

Since  Jacoby's  paper  appeared,  the  field  has  been  invaded  by  many 
workers,  who  have  examined  practically  everj^  tissue  in  the  body,  and 
found  that  all  possess  the  power  of  self-digestion;  or,  in  other  words, 
proteases  are  present  in  every  cell  in  the  hodij}  The  rate  of  digestion 
is  very  different  in  different  organs,  however,  liver  digesting  rapidly 
while  brain  and  muscle  tissue  digest  much  more  slowly,  aud  the  auto- 
lytic  activity  varies  under  different  conditions;^  thus,  fever  causes  a 
great  increase  in  the  proteolytic  activity  of  the  muscles.^*'  The  char- 
acter of  the  antiseptic  used  modifies  greatly  the  rate,  salicylic  and  ben- 
zoic acids  giving  the  most  rapid  autolysis,  while  of  non-acid  antiseptics 
toluene  is  perhaps  the  least  inhibitory.  One  of  the  most  important 
factors  in  modifying  the  rate  of  autolysis  is  the  H-ion  cencen- 
tration  developing  in  the  tissues. ^^  Acidity  acts,  partly,  at  least,  by 
so  modifying  the  substrate  that  the  enzj^mes  can  attack  it,  and  a  very 
small  excess  of  acid  will  destroy  the  enzymes;  Bradley^'-  estimates  this 
destructive  acidity  at  about  that  concentration  of  H-ions  which  is 
indicated  by  methyl  orange  and  Congo  red,  the  maximum"  autolysis 
being  obtained  with  an  acidity  at  about  pH  =  6.00.  A  reaction 
approximating  that  of  blood  (pH  =  7.4  —  7.8)  reduces  autolysis  to  a 
minimum.     A   latent   period  has  been  observed  before  autolysis  in 

«Zeit.  physiol.  Chem.,  1907  (53),  320. 

^  Jour.  Biol.  Chem.,  1910  (8),  61. 

^  Except,  perhaps,  the  red  corpuscles  (Pincussohn  and  Roques,  Biochem.  Zeit., 
1914  (64),  1). 

*  Concerning  autolysis  of  skin,  see  Sexsmith  and  Petersen,  Jour.  Exp.  Med. 
1917  (27),  273. 

'"  Aronsohn  and  Blumenthal,  Zeit.  klin.  Med.,  1908  (65),  1.  Striated  muscle 
autolyzes  much  less  rapidly  than  cardiac  and  unstriated.  (Bradley,  Proc.  Am. 
Soc.  Biol.  Chem.,  1918  (33),  xi). 

"  See  Morse,  Jour.  Biol.  Chem.,  1916  (24),  163. 

'Uour.  Biol.  Chem.,  1915  (22),  113;  1916  (25),  261. 


78  ENZYMES 

vitro  seems  to  begin,  part  of  which  time  may  be  occupied  in  the  develop- 
ment of  sufficient  acidity  to  permit  of  autolysis,  although  Bradley's^^ 
results  indicate  that  it  can  be  accounted  for  largely  by  the  time  re- 
quired for  proteolysis  to  proceed  far  enough  to  be  detected  by  chemical 
means.  Dernby^'*  finds  that  in  several  tissues  studied,  including 
leucocytes,  there  are  two  intracellular  proteases,  one  resembling  pepsin 
in  carrying  digestion  only  to  the  peptone  stage  and  in  requiring  an 
acid  medium,  optimum  pH  =  3.5;  the  other  resembling  ereptase, 
splitting  only  peptones  and  peptids  into  amino-acids,  with  optimum 
reaction  pH  =  7.8,  and  inhibited  by  acid  reaction.  Autolysis  of  tis- 
sues proceeds  furthest  in  a  pH  range  between  5  and  6,  presumably 
because  in  this  condition  both  enzymes  can  act.  From  these  facts  it 
is  evident  that  quantitative  studies  of  rates  of  autolysis  are  valueless  if  the 
H-ion  Tconcentration  is  not  taken  into  consider atio7i. 

The  cleavage  products  resulting  from  tissue  autolysis  seem  to 
contain  a  much  larger  proportion  of  the  nitrogen  in  the  form  of 
ammonia  and  its  compounds  than  is  the  case  with  simple  tryptic 
digestion,  because  of  the  presence  of  deaminizing  enzymes  which  split 
the  NH2  groups  out  of  the  amino-acids  and  purines.  According  to 
Bostock^^  the  greater  the  acidity  the  less  NH3  is  formed.  It  is  quite 
probable  that  in  tissue  autolysis  several  intracellular  enzymes  are  in 
action  which  may  not  be  present  in  pancreatic  or  gastric  juice;  for  ex- 
ample, in  the  liver  is  an  enzyme,  arginase,  which  splits  the  urea  radical 
out  of  the  arginine  of  the  proteins  (Kossel  and  Dakin^''),  and  the 
enzymes  which  disintegrate  purines  are  also  absent  from  the  digestive 
juices.  On  the  whole,  however,  the  products  are  quite  similar  to  those 
obtained  by  tryptic  digestion.  To  give  a  concrete  example,  Dakin^^ 
detected  in  the  products  of  autolysis  by  the  kidney  in  acid  solution,  the 
following  substances:  Ammonia,  alanine,  a-aminovalcrianic  acid,  leu- 
cine, a-pyrollidine  carboxylic  acid,  phenylalanine,  tyrosine,  lysine,  histi- 
dine,  cystine,  hypoxanthine,  and  indole  derivatives,  including  probably 
tryptophane.^^  The  cleavage  of  simple  peptids  by  different  tissues 
shows  characteristic  differences,  the  distribution  of  the  enzj-me  which 
splits  glycyl-tryptophane  having  been  most  studied.  During  life  the 
cells  retain  this  enzyme,  and  hence  it  appears  in  the  body  fluids  only 
when  the  tissues  are  being  rapidly  disintegrated  (Mandelbaum).^^ 

During  autolysis  the  changes  are  by  no  means  limited  to  the  pro- 
teins. Glycogen  is  split  into  glucose  very  early,  and  the  sugar  under- 
goes further  changes.     Fats  are  also  split  by  the  lipase,  fatty  acids 

"  Jour,  liiol.  Chcm.,  191G  (25),  3G3. 
!■•  .lour.  Biol.  Chem.,  1918  (35),  179. 
>5  Biochein.  Jour.,  1912  (6),  388. 
>6  Zeit.  physiol.  Chcm.,  1901  (42),  181. 
iMour.  of  PhysioloKY    1903  (30),  84. 

18  The  results  of  autolysis  by  (iilTcrcnt  tissues  are  said  to  be  quite  dissimilar. 
See  Kashi\val)ara,  Zeit.  i)hysiol.  Chcm.,  1913  (85),  IGl. 
'9  Miinch.  med.  Woch.,  1914  (01),  401. 


PRINCIPLES  OF  AUTOLYSIS  79 

being  found  in  iiutolyzod  organs.  Reducing  substances  appear,  and 
as  before  mentioned,  numerous  volatile  fatty  acids  are  said  to  be 
produced.  ^Vluch  doubt  exists  concerning  the  supposed  formation  of 
volatile  fatty  acids  and  gasses  during  autolysis  since  it  was  shown  by 
Wolbach,  Saiki  and  Jackson-"  that  anaerobic  bacteria  are  almost  in- 
variably present  in  aseptically  removed  dog  livers,  for  control  of  auto- 
lysis by  anaerobic  cultures  has  seldom  been  carried  out.  However, 
there  is  much  evidence  that  lactic  acid  is  formed,  and  perhaps  par- 
tially destroyed,  in  autolysis  (Tiirkel,^^  Ssobolcw^^).  Carefully  con- 
trolled experiments  by  Lindcmann-^  seem  to  show  that  even  in  the 
absence  of  bacteria,  autolyzing  liver  and  heart  can  produce  volatile 
acids,  CO2  and  hydrogen.  The  increase  in  fat  described  by  some 
authors  is  probably  only  apparent,  and  due  rather  to  the  liberation  of 
the  fat  from  its  combination  with  the  proteins  so  that  it  is  free  and 
not  "masked,"  as  in  normal  organs.^''  Lecithin  is  decomposed, 
yielding  choline,  but  cholesterol  remains  unchanged  except  for  some 
hydrolysis  of  cholesterol  esters. ^^  Creatine  is  changed  to  creatinine 
in  autolyzing  muscle,  and  apparently  both  are  formed  in  autolysis  of 
blood  and  liver. -^ 

The  nucleo-proteins  seem  to  be  attacked  by  the  autolytic  enzymes, 
as  the  purine  bases  are  prominent  among  the  products  of  autolysis, 
and  in  quite  different  proportions  from  those  obtaining  in  digestion 
of  the  same  tissues  by  other  means.  Apparently  autolytic  enzymes, 
like  trypsin,  attack  the  protein  group  of  the  nucleoproteins,  liberating 
the  nucleic  acids.  These  in  turn  are  attacked  by  specij&c  enzymes, 
nucleases,'^''  which  liberate  the  purine  bases,  which  are  further  decom- 
posed by  specific  enzymes,  guanase,  adenase,  etc.     (See  Chap,  xxiii). 

It  is  improbable  that  the  intracellular  enzymes  are  merelj^  pan- 
creatic enzymes  taken  out  of  the  blood  by  the  cells,  because  of  the 
differences  previously  cited;  furthermore,  Matthes^^  found  that  the 
liver  retained  its  autolytic  power  after  the  pancreas  had  been  extir- 
pated (in  dogs),  and  that  the  autolytic  degeneration  of  cut  peripheral 
nerves  went  on  just  the  same,  indicating  that  the  autolytic  enzj^mes 
do  not  owe  their  origin  to  the  pancreas. 

Whenever  tissues  are  disintegrated  in  any  considerable  quantities, 
as  after  extensive  burns,  peptolytic  enzymes  become  demonstrable  in 

20  Jour.  Med.  Res.,  1909  (21),  267. 

2»  Biochem.  Zeit.,  1909  (20),  431. 

"  Ibid.,  1912  (47),  367.  See  also  v.  Stein  and  Salkowski,  Biochem.  Zeit.,  1913 
(40),  486. 

"Zeit.  f.  Biol,  1910  (55),  36. 

2^  See  Krontowski  and  Poleff,  Beitr.  Path.  Anat.,  1914  (58),  407. 

"Corper,  Jour.  Biol.  Chem.,  1912  (11),  37;  Kondo,  Biochem.  Zeit.,  1910  (27), 
427. 

26  Myers  and  Fine,  Jour.  Biol.  Chem.,  1915  (21),  583;  Hoagland  and  McBrvde, 
Jour.  Agric.  Res.,  1916  (6),  535. 

"Sachs,  Zeit.  physiol.  Chem.,  1905  (46),  337;  Jones,  ibid.,  1903  (41),  101, 
and  1906  (48),  110. 

28  Arch.  f.  exp.  Path.  u.  Pharm.,  1904  (51),  442. 


80  ENZYMES 

the  blood  and  urine,  and  presumably  these  are  related  to  the  cell 
autolysis. 2^  They  are  noticeably  increased  in  most  infectious  diseases 
in  which  the  reaction  between  the  body  defenses  and  the  infecting 
organism  takes  place  in  the  blood  stream  (Falls). ^^  Also  in  the  pre- 
mortal state  a  similar  increase  in  peptolytic  enzyme  in  the  serum  is 
associated  with  a  high  non-protein  nitrogen  figure  for  the  serum. ^^ 
The  relation  of  the  autolytic  enzymes  to  the  increased  proteolytic 
power  of  serum  in  pregnancy,  as  evidenced  in  the  Abderhalden  reaction 
{q.v.)  has  not  yet  been  determined, ^^  ^^t  Falls  finds  evidence  of  their 
correlation.^"  Blood  proteases  are  also  increased  in  pregnancy. 
They  bear  no  constant  relation  to  the  leucocyte  count.  Autodigestion 
of  serum  is  normally  prevented  by  the  presence  of  a  specific  antienzyme, 
which  latter  can  be  inhibited  by  chloroform  and  various  saturated 
monovalent  ketones  and  alcohols  (Yamakawa).^^ 

Influence  of  Chemicals  on  Autolysis. — As  a  general  rule  the  addition  of  anti- 
septics to  tissues  to  prevent  bacterial  action  reduces  the  rate  of  autolysis,  but 
as  most  of  the  results  of  "aseptic"  autolysis  so  far  reported  are  open  to  question, 
there  is  a  reasonable  doubt  as  to  just  how  much  depression  of  autolysis  there  is. 
Yoshimoto^^  finds  that  of  the  antiseptics  ordinarily  used,  salicjdic  acid,  boric 
acid,  and  mustard  oil  (one-eighth  saturated  solution)  permit  the  greatest  auto- 
lysisj  but  it  is  probable  that  the  acidity  of  the  first  two  aiitiseptics  plays  an  im- 
portant part,  hence  the  value  of  the  results  obtained  in  autolysis  with  these  acids 
is  questionable.  However,  sodium  salicylate  and  benzoate  are  said  to  favor 
autolysis  (Laqueur).^^  Toluene  seems  to  interfere  much  less  with  autolj'sis 
than  chloroform  or  thymol  (Benson  and  Wells^*^),  and  bromides  are  less  harmful 
than  toluene  (Laqueur).  Toluene  vapor,  acting  on  solid  aseptic  tissues,  seems 
to  cause  more  depression  of  autolysis  than  is  usually  observed  in  autolysis  in 
solution.  ^^  Dorothy  Court^*  found  the  only  satisfactory  antiseptics  to  be  chloro- 
form, formaldehyde,  benzoic  and  salicj'lic  acids,  and  HNC;  she  emphasizes  the 
fact  that  for  different  sorts  of  materials  the  different  antiseptics  give  variable  re- 
sults, so  that  the  antiseptic  used  should  be  selected  with  reference  to  the  material. 
Autolysis  proceeds  rapidly  in  weak  ethyl  alcohol,  5  per  cent,  being  the  minimum 
strength  that  will  prevent  putrefaction;  for  complete  suppression  of  autolysis  by 
alcohol  the  strength  must  be  at  least  90  per  cent,  net,  after  allowing  for  the  water 
content  of  the  tissues  (Wells  and  Caldwell). ^^ 

Certain  inorganic  substances  in  proper  concentrations  have  been  reported  as 
increasing  the  rate  of  autolysis  [mercury^"  and  silver, ■'^   (colloidal^-  or  salts)], 

29  See  Pfeiffer,  Miinch.  med.  Woch.,  1914  (61),  1099,  1329. 

3"  Jour.  Infect.  Dis.,  1915  (16),  466;  also  Petersen  and  Short,  Jour.  Infect. 
Dis.,    1918   (22),    147. 

"  See  Schulz,  Miinch.  med.  Woch.,  1913  (60),  2512;  Mandelbaum,  ibid.,  1914 
(61),  461. 

32  See  Sloan,  Amer.  Jour.  Physiol.,  1915  (39),  9. 

33  Jour.  Exp.  Med.,  1918  (27),  689. 

34  Zeit.  physiol.  Chem.,  1908  (58),  341. 
36Zeit.  physiol.  Chem.,  1912  (79),  38  and  65. 

38  Jour.  Biol.  Chem.,  1910  (8),  61. 

3' Cruickshank,  Jour.  Path,  and  Bact.,  1911  (16),  167. 
38Proc.  Roy.  Soc,  Edinburgh,  1912  (32),  251. 

39  Jour.  Biol.  Chem.,  1914  (19),  57. 
"Truffi,  Biochem.  Zeit.,  1910  (23),  270. 
«  Izar,  ibid.,  1909  (20),  249. 

••2  The  accelerating  influence  of  colloidal  metals  is  denied  by  Bradley,  Proc. 
Amer.  Soc.  Biol.  Chem.,  1918  (33),  xi. 


PRINCIPLES  Op  AUTOLYSIS  SI 

yellow  pho.splioius,"  iodides/'  arsenic,'"  CaClo/'"'  salts  of  Kc,  Mr,  and  cobalt/' 
as  well  as  salts  of  selenium,  tclhiriuin,"  and  manganese, ■*'  colloidal  sulfur'"  but  not 
colloidal  carbon."  The  favorable  concentrations  of  these  metals  are  very  low;  thus 
the  optimum  proportion  of  arsenic  is  0.007  milli^;rams  i)er  1  Km.  tissue,  while  0.04 
mg.  inhibits  autolj'sis.  CO2  increases  and  oxyncn  decreases  autolysis"  in  vitro 
(Laqueur).  There  is  disagreement  as  to  whether  radium  rays  augment  autolysis.*' 
Injection  of  iodids  into  animals  is  said  to  increase  the  postmortem  autolysis  of 
their  tissues  (Stookey,  Kepinow),  as  also  do  iron  salts,'"'  while  large  doses  of 
salicylates  decrease  it  (Laqueur).  Morse"  attributes  the  accelerating  action 
of  iodin  and  bromin  to  increased  acidity  from  formation  of  halogen  acids,  and 
Bradley*^  finds  evidence  that  most  inorganic  salts  that  stimulate  autolysis  act 
by  increasing  Fl-ion  concentration.  Addition  of  tuberculin  to  tissues  at  first 
delays  and  tiien  increases  the  autolysis  (Pesci^"*),  and  diphtheria  toxin  in  small 
amounts  increases  autolysis  (Barlocco,"  Bertolini**),  neutralization  by  anti- 
toxin not  preventing  this  effect.  Lipoids  also  accelerate  autolysis  (Satta  and 
Fasiani").  According  to  Soula*"  narcotic  poisons  decrease,  and  convulsive 
poisons  increase  the  rate  of  autolysis  of  nervous  tissue.  Glucose  in  one  per  cent, 
concentration  decreases  autolysis,  and  this  may  be  related  to  the  ''protein-sparing 
action  of  carbohydrates."*'  E.xtracts  of  various  ductless  glands,  or  removal  of 
these  glands  from  animals,  seem  to  have  but  slight  effect  on  autolysis.*- 

In  considering  the  foregoing  statements  allowance  must  be  made  for  the  fact 
that  in  most  of  the  work  cited  there  has  been  no  proper  consideration  of  H-ion 
concentration  in  the   autolyzing  mixtures. 

Relation  of  Autolysis  to  Metabolism 

It  having  been  shown  that  proteases  are  present  in  all  cells,  the 
next  question  to  be  considered  is,  do  they  act  only  to  destroy  tissues 
after  death,  or  are  they  of  importance  in  metabolism?  Since  it  is 
presumably  necessary  for  proteins  to  be  split  into  diffusible  and  easily 
oxidized  forms  in  order  that  they  may  enter  the  cell,  and  be  built  up 
into  the  cell  proteins,  or  be  decomposed  with  the  liberation  of  energy, 
the  autolytic  proteases  ma}^  be  assumed  to  be  of  prime  importance  in 
protein  metabolism;  but  to  prove  it  is  another  matter.     Jacoby  found 

"  Saxl,  Hofmeister's  Beitr..  1907  (10).  447;  Virchow's  Arch.,  1910  (202),  149. 

^  Kepinow^  Biochem.  Zeit.,  1911  (37),  238.  Kaschiwabara,  Zeit.  phvsiol.  Chem., 
1912   (82),  425.     Not  confirmed  by  Albrecht,  Jour.  Biol.  Chem.,  1919  (41),  111. 

^5  Izar,  Biochem.  Zeit.,  1909  (21),  46;  Laqueur  and  Ettinger,  Zeit.  physiol. 
Chem.,  1912  (79)    1. 

«Briill,  Biochem.  Zeit.,  1910  (29),  408. 

^"Preti,  Zeit.  phvsiol.  Chem.,  1909  (GO),' 317;  PoUini,  Biochem.  Zeit.,  1912 
(47),  396. 

"  Fasiani,  Arch.  sci.  med.,  1912  (36),  436. 

^9  Bradley,  Jour.  Biol.  Chem.,  1915  (21),  209;  1915  (22),  113. 

5"  Faginoli,  Biochem.  Zeit.,  1913  (56),  291. 

*'  Izar  and  Patane,  ibid.,  p.  307. 

^'  M.  Morse  found  oxvgen  without  effect  on  autolysis.  Biochem.  Bullet.,  1915 
(5),  143. 

"  See  Loewenthal  and  Edelstein,  Biochem.  Zeit.,  1908  (14),  485;  Brown,  Arch. 
Int.  Med.,  1912  (10),  405. 

•■*<  Kottmann,  Zeit.  exp.  Path.,  1912  (11),  355. 

"Jour.  Biol.  Chem.,   1915  (22),  125. 

5«Cent.  f.  Bakt.,  1911  (59),  71  and  186. 

"Cent.  f.  Bakt.,  1911  (60),  43. 

s' Biochem.  Zeit.,  1913  (48),  448. 

59Berl.  klin.  Woch     1910  (47),   1.500. 

^oCompt.  Rend.  Soc.  Biol.,  1913  (73),  297. 

"  Shaffer,  Proc.  Soc.  Biol.  Chem.,  1915  (8),  .xlii. 

*\Izar  and  Fagiuoli,  Sperimentale,  1916  (70),  265. 
6 


82  ENZYMES 

that  if  he  hgated  off  a  portion  of  the  hver  and  let  it  remain  in  situ 
in  the  animal  the  necrotic  tissues  showed  an  accumulation  of  leucine, 
tyrosine,  and  other  cleavage  products  of  the  proteins,  which  suggested 
that  these  same  bodies  are  being  formed  in  the  liver  constantly,  but 
that  they  are  as  constantly  removed  from  the  normal  organs  by  the 
circulating  blood,  or  are  undergoing  further  alterations  which  cease 
when  the  circulation  is  checked.  The  influence  of  various  chemicals 
upon  nitrogen  elimination  seems  to  correspond  to  their  effect  on  auto- 
lysis (Izar,^^  Laqueur^^).  Also,  the  histologic  changes  of  starvation 
are  similar  in  many  respects  to  those  of  autolysis  (Casa-Bianchi^^). 
Among  other  observations  possibly  bearing  on  the  same  question  are 
those  of  Hildebrandt,^^  who  found  that  autolysis  in  the  functionating 
mammary  gland  is  much  more  active  than  in  the  resting  gland;  and 
of  Schlesingeir,^^  who  found  that  autolysis  was  at  its  maximum  (in 
rabbits)  in  new-born  animals,  decreasing  rapidly  in  the  first  few 
months  of  life,  and  that  in  conditions  associated  with  emaciation 
the  rate  of  autolysis  varied  directly  with  the  degree  of  emaciation. 
Wells^^  sought  for  a  possible  influence  on  autolysis  by  thyroid  extract, 
which  increases  protein  metabolism,  but  could  demonstrate  none 
in  vitro;  Schryver,^^  however,  reported  that  autolysis  was  more  rapid 
in  the  liver  of  dogs  fed  thyroid  extract  for  some  days  before  death  than 
it  was  in  control  animals.  The  results  of  the  former  observer,  but  not 
those  of  the  latter,  have  been  confirmed  by  Morse. ^^ 

Defense  of  the  Cells  Against  their  Autolytic  Enzymes 

The  question  of  why  the  autolytic  ferments  do  not  destroy  the 
cells  until  after  death  is  a  revival  of  the  old  problem  of  ''why  the 
stomach  does  not  digest  itself,"  and  the  answer  that  satisfies  some  is 
that  dead  protoplasm  is  essentially  different  from  living  protoplasm. 
More  specific  replies  are  suggested  by  Wiener's  studies  on  the  relation 
of  the  reaction  of  the  tissues  to  their  autolysis.  He  found  that  auto- 
lysis does  not  begin  in  an  organ  until  the  original  alkalinity  is  neutra- 
lized by  the  acids  which  are  formed  in  all  dead  and  djnng  cells. ^'  If 
enough  alkali  is  added  to  the  material  from  time  to  time  to  neutralize 
the  acidity  as  it  develops,  autolysis  docs  not  take  place.     Although 

«'Internat.  Beitr.  Erniihrungstor.,   1910  (1),  287. 

"  Zeit.  physiol.  Cheni.,  1912  (79),  1  et  seq. 

"  Frankfurter  Zeit.  Pathol.,   1909  (3),  723. 

""  Hofmeister's  Beitrilge,  1904  (5),  463;  see  also  Grinimcr,  Hiochcni.  Zeit.,  1913 
(53),  429. 

"  Hofmeister's  Reitr.,   1903   (4),  87. 

"*  Amer.  Jour,  of  Physiol.,  1904  (11),  351;  eorrohorated  bv  Kottiiuinn,  Zeit. 
klin.    Med.,   1910   (71),  '•.m). 

«»  Jour,  of  Physiol.,  1905  (32),  159. 

'ojour.  Biol.  (Jhem.,  1915  (22),  125. 

"  ()])io  (toe  cU.)  found,  liowevcr,  llial  avitolysis  of  leucocytes  was  more  rapid 
in  an  alkaline  inediuiii.  Doeliez  (.lour.  \<]\]).  Med.,  1910  (,12),  tHKi)  stnt(>s  that  liver 
also  contains  an  enzyme  active  in  an  alkaline  meiliiun,  hut  which  exists  as  an 
inactive  zymogen  until  activated  by  acids.     See  also  Dernhy." 


DEFENSE  AGAINST  AUTOLYSIS  83 

the  spleen  contains  an  enzyme  digest inj!;  in  alkaline  solution,"  and 
another  which  acts  best  in  weak  acids,  the  latter  appears  more 
proniinontly  under  ordinary  conditions  because  the  spleen  and  the 
blood  contain  antibodies  which  check  the  enzyme  that  acts  in  alka- 
line solutions,  while  acids  destroy  this  antibody  (Hedin).^^  Organic 
acids  are  formed  in  autolysis  of  the  tissues,  and  the  latent  period  be- 
tween the  time  of  the  removal  of  an  organ  from  the  body  and  the  ap- 
pearance of  autolysis  may  be  explained  partly  by  the  time  recjuired 
for  the  neutralization  or  alkalinity.  Bradley'^  has  also  obtained  evi- 
dence that  the  acid  renders  the  substrate  susceptible  to  digestion  by  the 
proteases.  Dernby's^^  demonstration  of  the  existence  of  pepsin-like 
and  erepsin-like  enzymes  suggests  that  there  must  be  developed 
enough  acidity  to  permit  the  peptase  to  form  peptones  before  the 
ereptases  can  begin  their  further  cleavage.  Maximum  autolysis  is 
known  to  occur  when  tissues  are  first  made  acid  and  then  neutralized 
or  slightly  alkalinized  (Hedin) .  The  old  observation  that  rigor  mortis 
disappears  most  rapidly  in  muscles  that  have  been  exhausted  just 
before  death  is  probably  explained  by  the  greater  amount  of  acid  in 
such  muscles.  If  we  imagine  that  autolysis  is  limited  to  periods 
when  the  cells  have  an  acid  reaction,  however,  we  limit  the  range  of 
usefulness  in  the  living  cell  to  a  minimum,  since  during  life  the  tissue 
fluids,  and  presumably  the  cell  contents,  are  preponderatingly  alka- 
line. The  control  of  autolysis  by  maintenance  of  a  low  H-ion  concen- 
tration is  undoubtedly  an  important  factor,  for  Bradley  found  that  a 
reaction  equal  to  that  of  blood  almost  completely  inhibits  autolysis, 
while  the  degree  of  increased  H-ion  concentration  that  may  develop  in 
local  asph3^xia,  or  after  death,  produces  optimum  conditions  for 
autolysis. 

Still  another  possible  defense  of  the  Hving  cells  may  be  found  in 
the  existence  of  specific  antienzymes.  Just  as  the  serum  contains  anti- 
trypsin, so  it  seems  to  contain  substances  antagonistic  to  the  autolytic 
enzymes.  Levene  and  Stookey  found  that  tissue  juices  show  a  resist- 
ance to  digestion,  Yamakawa^^  found  that  serum  autolysis  is  prevented 
by  an  antienzyme,  and  Opie  found  that  the  serum  of  inflammatory 
exudates  retards  the  action  of  the  autolytic  enzymes  that  are  con- 
tained within  the  leucocytes.  Serum  also  inhibits  autolysis  of  the 
tissues,  so  it  is  probable  that  continuance  of  the  circulation  may  pro- 
vide antibodies  to  the  tissues  to  hold  the  intracellular  enzymes  in 
check,  possibly  without  interfering  with  their  action  on  other  pro- 
teins than  those  of  the  cell  structure.''*     (See  Antienzymes.)     It  is 

^2  Morse  (Jour.  Biol.  Chem.,  1917  (31),  303)  considers  this  enzyme  to  be  heter- 
olj'tic,  derived  from  the  white  cells. 

"  Festschrift   f.    Hammarsten,    Upsala,    1906. 

'••According  to  Guggenheimer  (Deut.  Arch.  khn.  Med.,  1913  (112),  248;  Dent, 
med.  Woch.,  1914  (40),  63),- the  serum  in  various  diseases  has  a  characteristic 
stimulating  or  inhibiting  effect  on  in  vitro  tissue  autolj'sis,  but  the  conditions  of 
such  experiments  are  so  complex  as  to  make  their  significance  doubtful. 


84  .  ENZYMES 

highly  probable  that  serum  cheeks  autolysis  at  least  in  part  by  virtue 
of  its  "buffer"  function,  which  interferes  with  the  development  of 
acidity.  Lack  of  oxygen  cannot  be  held  solely  responsible  for  auto- 
lysis, according  to  the  stuches  of  Morse, ^^  who  found  that  autolysis 
occurs  in  muscles  with  divided  nerves  but  intact  blood  supply.  Never- 
theless, reduced  blood  supply  results  in  increased  H-ion  concentration 
which  greatly  facilitates  autolysis,  and  it  cannot  be  denied  that  auto- 
lysis is  observed  chiefly  if  not  solely  in  asphyxiated  tissues. 

There  can  be  no  question  that  the  supply  of  food-stuff  is  of  essential 
importance  in  determining  autolytic  changes,  for  it  has  been  found 
by  Conradi,^^  Rettger,^''  and  Effront'*  that  bacteria  and  yeasts  begin 
to  undergo  autolysis  when  they  are  placed  in  distilled  water  or  salt 
solution,  which  they  do  not  do,  to  any  such  extent  at  least,  when  in 
nutrient  media.  (In  this  way  it  has  been  found  possible  to  obtain 
the  intracellular  poisons  of  such  bacteria  as  tj^phoid  and  cholera.) 
Autolysis  is  not  marked  so  long  as  the  bacteria  are  supplied  with 
nourishment,  but  when  nutrient  material  is  lacking,  autolytic  decom- 
position is  no  longer  repaired  and  the  bacteria  disintegrate.  Pre- 
sumably the  changes  are  the  same  in  tissue  cells,  and  anemic  necrosis 
may  be  explained  in  this  wa}'.  Tissue  enzymes  are  also  capable  of 
digesting  bacteria  (Turro^^). 

Another  direction  in  which  the  key  to  the  action  of  these  enzymes 
may  be  sought  has  been  indicated  by  Jacoby,^*^  who  found  that  to  a 
certain  degree  the  autolytic  enzymes  of  each  organ  are  specific  for  that 
organ.  Liver  extract  will  not  split  lung  tissue,  although  it  will  split 
the  proteoses  that  are  formed  in  lung  autolysis,  possibly  because  these 
proteoses  are  less  specific  than  the  proteins  from  which  they  arise,  or 
perhaps  because  of  the  erepsin  the  extract  contains  (Vernon).  Leuco- 
cytic  proteases,  however,  seem  capable  of  splitting  foreign  proteins 
of  all  sorts.  Richet^^  states  that  the  protease  of  liver  tissue  does 
not  attack  either  muscle  tissue  or  liver  tissue  that  has  been  coagulated. 
Another  hypothesis  has  been  advanced  by  Fermi,*-  who  suggests 
that  the  protoplasm  of  living  cells  is  not  digested  because  its  structural 
configuration  is  such  that  the  enzymes  cannot  unite  with  it,  an  attract- 
ive but  practically  undemonstrable  idea. 

Lastly,  it  must  be  considered  that  at  least  to  some  extent  the  en- 
zymes exist  in  the  cells  in  their  inactive  zymogen  form,  and  per- 
haps are  changed  into  the  active  form  as  needed,  and  inhibited  or 
changed  back  again   wlien   their   work   is   temporarily   finished.     A 

"  Amer.  Jour.  Phvsiol.,  lUlf)  (liO),  147. 
'»  Deut.  mod.  Woi-li.,    l<)():}  (2«)),  20. 
"Jour.   Med.   Hcsciiroh.   n)04   (Ui),  79. 
'8  Bull.   Soc.   Cliiiii.,    HH).")   (;};{),  847. 
'»Cont.  f.  Bakt.,  1902  (:V2),  105. 
»«  llofineistcr's    Boitr.,    190:}    (;}),    440. 
8'  (Joiiipt.  Boiul.  Soc.   Biol.,   190:}  {'•>'■>),  tiot). 
82  Cent.   1'.    Bakl.,    1910   (■>(>),   .1."). 


AUTOLYSIS  ].\  JWTIIOIAHIKAL  I' HOC  ESSES  85 

rhythmical  chaiijic  of  tliis  nalui'c  might  be  imajiiiicd  as  occuniMg  and 
accounting  for  interaction  by  the  enzymes,  particiihirly  since;  rliythmi- 
cal  changes  in  metabolism  are  known  to  occur  {e.  g.,)  rhythmical  pro- 
duction of  carbon  dioxide  (Lyon^^),  and  enzyme  action  in  vitro  may 
show  rhythmic  variations  (Groll).^'* 

Autolysis  in  Pathological  Processes 

All  absor];)tion  of  dead  or  injured  tissues,  and  of  organic  foreign 
bodies,  seems  to  be  accomplished  by  means  of  digestion  by  the  enzymes 
of  the  cells  and  tissue  fluids.  We  may  distinguish  between  the  diges- 
tion brought  about  bj^  the  enzymes  of  the  digested  tissue  itself,  or 
autolysis,  and  digestion  by  enzymes  from  other  cells  or  tissue  fluids, 
or  hetcrolysis  (Jacoby).  Heterolysis  is  accomplished  particularly  by 
the  lecucocytes,  which  contain  ferments  capable  of  digesting  not  only 
leucocytic  proteins  but  apparentlj^  every  other  sort,^''  from  serum- 
albumin  to  catgut  ligatures.  The  heterolysis  may  be  intracellular 
when  the  material  to  be  digested  has  first  been  taken  up  by  the  cells 
(phagocytosis);  or  extra-cellular,  either  by  enzymes  normally  con- 
tained in  the  blood  plasma  and  tissue  fluids,  or  by  enzymes  liberated 
by  the  leucocytes  and  fixed  tissue  cells.  On  death  and  dissolution  of 
a  cell  the  intracellular  enzymes  are  released, ^^  but  it  is  not  known  to 
what  extent  the  enzymes  may  be  secreted  from  intact  living  cells.  As 
far  as  pathological  processes  show,  the  amount  of  liberation  of  en- 
Z3'mes  from  normal  cells  is  very  slight,  if  any,  and  the  digestive  en- 
zymes of  the  blood  plasma  seem  to  be  very  feeble,  but  this  is  perhaps 
because  they  are  largely  held  in  check  by  the  anti-enzymatic  substances 
of  the  serum.  Pathological  autolysis  and  heterolysis,  therefore,  are 
brought  about  chieflj'^  bj'  enzymes  liberated  from  dead  or  injured  cells. 
Bacteria,  however,  can  multiply  upon  a  medium  of  coagulated  protein, 
which  suggests  that  they  also  secrete  proteolytic  substances.  In 
pathological  conditions  digestion  of  degenerated  tissues  seems  usuallj'^ 
to  be  the  result  of  both  autolysis  and  heterolysis.  An  infarct  softens 
because  the  intracellular  enzymes  digest  the  dead  cells,  exactly  as 

«^  Science,    1904    (19),   350. 

8^  Nederl.  Tijdschr.  v.  Geneesk.,  1918  (1),  1085. 

8^  Manj^  authors  suggest  that  the  leucocytes  merely  carry  enzymes  from  one 
organ,  particularly  the  pancreas,  to  another,  and  that  these  enzymes  are  not 
formed  by  the  leucocyte  itself.  Opie  (Jour.  Exp.  Med.,  1905  (7),  759)  has  shown, 
however,  that  the  bone-marrow  contains  proteolytic  enzymes  which  are  like  those 
of  the  leucocytes  in  that  they  act  best  in  an  alkaline  medium,  whereas  the  auto- 
lytic  enzymes  of  the  lymphatic  glands  and  most  other  tissues  act  best  in  an  acid 
medium.  This  leaves  little  room  for  doubt  that  the  leucocj'tes  are  equipped 
with  their  characteristics  enzjmies  when  they  leave  the  bone-marrow,  and  that 
they  are  not  obtained  later  in  the  pancreas  or  elsewhere.  More  recently,  however, 
van  Calcar  (Pfliiger's  Archiv.,  1912  (148),  257)  has  revived  the  idea  of  the  origin  of 
leucocj'tic  enzymes  in  the  digestive  glands. 

8^  Peptolytic  enzymes  appear  in  the  urine  after  severe  superficial  burning,  pre- 
sumably coming  from  the  disintegrated  cells.  (Pfeiffer,  Miinch.  med.  ^^'och., 
1914  (61),  1329.) 


86  ENZYMES 

they  do  when  the  tissue  is  removed  from  the  body,  ground  up,  and 
put  in  the  incubator  under  toluene.  In  addition  leucocytes  wander  in, 
disintegrate,  and  their  liberated  enzymes  help  in  the  process,  as  also  do, 
to  a  less  degree,  the  enzymes  of  the  blood  plasma.  It  is  because  of  the 
heterolysis  by  leucocytic  enzymes  that  a  septic  infarct  becomes 
softened  so  much  more  rapidly  than  does  a  sterile  infarct,  and  by 
comparing  the  rate  of  softening  in  septic  and  aseptic  infarcts  we  see 
that  the  cellular  autolysis  is  a  very  slow  process  as  compared  to  the 
heterolysis  accomplished  by  the  leucocytes.  The  explanation  of  this 
may  lie  in  the  fact  that  most  intracellular  proteases  act  best  in  an 
acid  medium  (Wiener),  while  leucocytic  proteases  act  best  in  an 
alkaline  medium  (Opie),  and  the  infarcts  of  small  size  are  seeped 
through  by  alkaline  blood  fluids.  When  an  infarct  is  large,  we  find 
it  undergoing  central  softening  while  the  periphery  remains  firm;  this 
corroborates  our  hypothesis,  for  acids  are  developed  during  autolysis 
(Magnus-Levy),  which  at  the  periphery  are  neutralized  by  the  blood 
plasma,  so  that  only  at  the  center  is  autolysis  active.  The  inhibit- 
ing action  of  the  serum  also  has  a  similar  effect,  limiting  autolvsis  at 
the  periphery.  Necrotic  areas  of  any  kind  are  absorbed  b}''  similar 
processes. 

Apparently  all  varieties  of  cells  are  subject  to  autolysis  or  heterolysis 
whenever  they  are  killed  or  sufficiently  injured.  Involution  of  the 
uterus  probably  depends  upon  autolysis,  which  is  much  more  active 
in  the  puerperal  uterus  (Ferroni^^),  and  creatine  is  found  in  the  urine 
when  such  autolysis  occurs, ^^  although  A.  Morse^^  considers  this  to  be 
independent  of  the  uterine  autolysis.  Atrophy  may  be  looked  upon  as 
an  autolysis  in  the  normal  course  of  catabolism,  not  met  by  a  corres- 
ponding building  up  of  the  proteins,  but  M.  IVIorse^^  could  find  no 
evidence  that  the  atrophy  and  involution  of  the  tadpole  tail  is  ac- 
companied by  an  accelerated  autolysis.  The  solution  of  fibrin  by 
tissues,  fibrinolysis,  is  considered  to  be  distinct  from  tissue  autolysis 
by  Fleisher  and  Loeb.^''  In  atrophic  cirrhosis  the  fibrinolytic  activity 
of  the  blood  is  increased,  which  may  explain  the  hemorrhagic  tendencj' 
of  this  disease. ^^  In  the  case  of  septic  softening  the  action  of  the 
bacteria  needs  also  to  be  taken  into  consideration,  since  they  produce 
proteolytic  ferments,  but  their  effect  seems  to  be  relatively  small  as 
compared    with    leucocytic    digestion.^-      Intracellular    digestion    of 

*'  Arm.  di  Ostetrica  e  Ginecol.,  1906  (2),  553;  see  also  Sleinons,  Bull.  Johns 
Hopkins  IIosp.,  1914  (25),  195;  Arthur  Morse,  Jour.  Amer.  Med.,  Assoc,  1915 
(05),  l()i:i. 

8«  Shaffer,  Amer.  .Jour.  Physiol.    1908  (23),  1. 

89  Max  Morse,  Am.  Jour,  i'hysiol.,  1915  (30),  145. 

90  .Jour.  Biol.  Chcm.,  1915  (21),  477. 

91  Goodi)asture,  iiull.  .Johns  Hopkins  JTosp.,  1914  (25),  330. 

92  Tlic  enzymes  of  staphylococcus  are  nmcli  more  strongly  proteolytic  tlian  those 
of  streptococcals  (Knapj),  Zeit.  f.  Heilk.  (Chir.),  1902  (23),  230.)  which  may  he 
one  reason  why  tlie  latter  so  much  more  frequently  produces  lesions  without  suj)- 
puration  tlian  does  the  former. 


AUTOLYSIS  IN  PATHOLOGICAL  PROCESSES  87 

necrotic  tissue  by  leucocytes  seems  also  to  be  relatively  unimportant. 
Suppuration,  therefore,  must  be  considered  as  the  result  of  digestion 
of  dead  tissue  by  enzymes  derived  from  the  leucocytes,  the  plasma, 
the  bacteria,  and  the  destroyed  cells  themselves.  A  tubercle  does 
not  ordinarily  suppurate,  because  the  tubercle  bacillus  and  the  sub- 
stances it  produces  are  not  strongly  chemotactic,  and  hence  not 
enough  leucocytes  enter  the  necrotic  area  to  produce  a  digestive 
softening. 

The  products  of  autolysis  may  of  themselves  be  toxic;  albumoses 
and  peptones  certainly  are,  and  the  other  cleavage  products  are  prob- 
ably not  altogether  innocuous.  (See  "Autointoxication.")  Some  of 
the  symptoms  of  suppuration,  particularly  the  fever  and  chills,  have 
been  ascribed  to  the  autolytic  products  rather  than  to  the  bacterial 
poisons,  particularly  as  aseptic  suppuration  is  accompanied  by  fever. 
Jochmann^^  has  found  evidence  that  the  protease  of  leucocytes  can 
cause  fever  and  also  reduce  the  coagulability  of  the  blood.  The  work 
of  Vaughan  and  other  recent  students  of  the  reaction  to  foreign  pro- 
teins, shows  that  typical  fevers  can  be  produced  by  the  enzymatic 
disintegration  of  proteins  in  the  body.^'*  Degenerative  changes  in 
nervous  tissue  are  associated  with  autolytic  decomposition  of  the 
lecithin  (NolP^)  and  the  liberated  choline,  or  its  more  toxic  derivatives, 
may  be  a  source  of  intoxication.^^  In  all  conditions  associated  with 
autolysis,  such  as  resolving  pneumonic  exudates,  large  abscesses,  soften- 
ing tumors,  etc.,  albumoses  (and  peptones?)  may  appear  in  the  urine. 
Autolytic  products  may  also  be  hemolytic  (Levaditi^^),  and  they  may 
prevent  clotting  of  the  blood  (Conradi^^).  It  is  probable  that  among 
the  products  of  autolysis  are  bactericidal  substances, ^^  although  it  is 
doubtful  if  the  concentration  is  often  sufficient  for  them  to  be  of 
influence  except  in  well  walled  areas. 

There  is  also  much  evidence  that  after  extensive  traumatism, 
especially  as  observed  in  war,  the  products  of  the  tissue  autolysis  may 
be  responsible  for  serious  intoxication,  and  possibly  for  conditions 
interpreted  at  times  as  shock.  ^  The  observations  made  in  experimental 
anaphylaxis  suggest  that  it  is  especially  the  slightly  altered  proteins, 
perhaps  only  changed  in  their  colloidal  properties,  that  are  most  likely 
to  be  responsible  for  these  shock-like  intoxications.  However,  it  is 
also  possible  that  amines  derived  from  the  aminoacids  may  be  of 

9'Virchow's  Arch.,  1908  (194),  342. 

9^  See  Vaughan,  "Protein  Split  Products,"  Philadelphia,  1913. 
«  Zeit.  phvsiol.  Chemie,  1899  (27),  380. 
9«  See  Halliburton,  Ergebnisse  der  Physiol.,  1904  (4),  24. 

"  Ann.  d.  I'lnst.  Pasteur,  1903  (17),  187;  also  Fukuhara,  Zeit.  f.  exp.  Path.  u. 
Pharm.,  1907  (4),  658. 

98  Hofmeister's  Beitr.,  1901  (1),  136. 

99  See  Bilancioni,  Arch,  farmacol.,  1911  (11),  491. 

1  Delbet,  Bull.  Acad.  Med.,  1918  (80),  13;  Cannon,  Compt.  Rend.  Soc.  Biol., 
1918  (81),  850;  Turck,  Med.  Record,  June  1,  1918. 


88  ENZYMES 

importance  in  producing  shock  whenever  tissues  are  injured.^  Methyl 
guanidine  may  also  be  formed  from  disintegrating  tissues  and  has  con- 
siderable toxicity. 

Work  has  been  reported  upon  autolytic  processes  in  a  number  of 
pathological  conditions,  which  may  be  discussed  briefly  as  follows: 

Exudates. — The  presence  of  leucine,  tyrosine,  proteoses,  and  pep- 
tones in  pus  has  been  known  for  many  years,  and  the  reason  for  their 
appearance  is  now  clear.  Muller,^  many  years  ago,  observed  that 
purulent  sputum  digested  fibrin,  but  that  non-purulent  sputum  did 
not  have  this  property.  Achalme^  found  that  pus  would  dissolve 
gelatin,  fibrin,  and  egg-albumen.  Ascoli  and  Mareschi^  detected 
autolysis  in  sterile  exudates  obtained  experimentally.  Umber^  found 
that  ascitic  fluid  exhibited  autolytic  changes,  which  observation 
could  not  be  confirmed  by  Schiitz^  in  pleural  exudates  and  ascitic 
fluids.  Zak^  found  that  autolysis  was  inconstant  in  various  exudates. 
The  differences  in  these  results  are  explained  by  Opie's^  observation 
that  in  experimental  inflammatory  exudates  the  leucocytes  are  capable 
of  marked  autolysis,  whereas  the  serum  contains  an  antibody  which 
holds  this  autolysis  in  check;  if  the  antibody  is  destroyed  by  heat,  then 
the  serum  proteins  are  also  digested  by  the  leucocytic  enzymes.  This 
antibody  seems  to  be  contained  normally  in  the  albumin  of  the 
blood-serum.  In  old  exudates  the  antibodies  are  decreased,  and  auto- 
lysis then  occurs,  explaining  the  variable  results  of  Umber,  Schiitz 
and  Zak.  The  intracellular  proteases  of  the  polynuclear  leucocytes 
act  best  in  an  alkaline  medium;  those  of  the  mononuclears  in  acid 
medium.  If  the  proportion  of  serum  to  leucocytes  is  high,  then  there 
is  no  autolysis,  as  in  serous  exudates;  but  if  the  leucocj^tes  are  abun- 
dant, then  the  antibody  is  overcome  and  we  get  autoh'sis,  as  in  ordinary 
suppurative  exudates.  Animals  with  but  little  protease  in  their 
leucocytes  (e.  g.,  rabbits),  do  not  ordinarily  produce  a  liquid  pus  (Opie). 
Exudates  produced  by  bacterial  infection  also  seem  to  possess  the 
properties  above  described.  Galdi^"  found  autolysis  greater  in  exu- 
dates than  in  transudates,  but  observed  no  constant  relation  between 
the  number  of  leucocytes,  or  the  amount  of  chlorides,  and  the  rate  of 
autolysis.  All  exudates,  according  to  Lenk  and  Pollak,^^  contain 
enzymes  splitting  glyeyl-glycinc  (peptolytic  enzymes);  the  most  active 

2  See  Abel  and  Kubota,  Jour.  Pharm.  Exp.  Ther.,  1919  (13),  243. 

3  Kossel,  Zeit.  f.  klin.  Med.,  18S8  (13),  149. 
^  Compt.  Rend.  Soc.  Biol.,  1899  (51),  568. 

6  See  Maly's  Jahresbericlit,   1902  (32),  5(38. 
6  Munch,  nied.  Woch.,   1902  (49),   11(59. 
'Cent.  f.  inn.  Med.,  1902  (23),  ll(il. 

8  Wien.  klin.  Woch.,  1905  (18),  37(j. 

9  Jour,   of  Exper.  Med.,  1905  (7),  310  and  759;  190(i  (8),  410  and  530;  1907 
(9),  207,  391  and  414;  also  a  full  review  in  Arcii.  Int.  Med.,  1910  (5),  541. 

'0  See  Folia  Ilcinat.,   1905  (2),  529. 

'»  Dcut.  Arcli.  klin.  Med.,  1913  (109),  350;  See  also  Wiener,  liiocliein.  Zeit., 
1912  (41),  149;  Al!iiid<'ll)auiii,  Miinch.  lued.  Woch.,  1914  ((il),  4()1. 


AUTOLYSIS  IN  PATHOLOGICAL  PROCESSES  89 

exudates  are  tliose  of  cancer  and  tuberculosis,  tlie  least  active  are 
passive  congestion  fluids;  pleural  exudates  contain  more  active  enzymes 
than  peritoneal  exudates  of  .similar  character. 

Knapp^-  holds  that  in  pus  the  cocci  and  the  enzymes  they  produce 
are  responsible  for  much  of  the  digestion.  Pus  cells  alone  do  not 
undergo  digestion  so  rapidly  as  when  bacteria  are  present,  and  di- 
gestion is  more  rapid  if  the  bacteria  are  alive  than  when  inhibited  or 
killed  by  antiseptics.  Streptococcus  is  almost  inactive,  staphylococcus 
is  quite  active,  and  B.  coli  still  more  so.  However,  pus  corpuscles 
free  from  bacteria  are  highly  proteolytic,  causing  digestion  in  serum 
plates  in  dilutions  of  1-700  (Jochmann).  Knapp  could  find  no  rela- 
tion between  the  autolytic  power  of  the  pus  and  the  severity  of  the  in- 
fection from  which  it  resulted.  A  constant  constituent  of  pus  is 
d-lactic  acid, ^2  ^nd  it  increases  during  autolysis;  this  may  well  modify 
the  rate  of  autolysis  of  pus.  (See  also  the  discussion  of  the  "Chem- 
istry of  Pus,"  Chap,  xi.) 

Proteolytic  Enzymes  of  the  Leucocytes.^^ — By  the  introduction  of 
the  plate  method  of  testing  the  proteolytic  activitj^  of  leucocytes, 
Miiller  and  Jochmann  brought  the  study  of  this  particular  vital 
activity  into  the  range  of  clinical  laboratories,  and  aroused  much 
general  interest  in  what  had  previously  concerned  only  a  few  pathol- 
ogists, especially  E.  L.  Opie.  The  principle  is  that  of  permitting 
the  leucocj^tes  or  other  cells  to  act  upon  a  blood  serum  plate  at  a  tem- 
perature of  55°,  which  prevents  bacterial  action  but  permits  the  pro- 
teolytic enzymes  of  the  cells  to  digest  the  coagulated  serum,  forming 
depressions  in  the  surface  ("Dellbildung").  This  proteolytic  activity 
is,  of  course,  heterolysis  rather  than  autolysis.  Many  modifica- 
tions of  this  method  have  been  introduced  (such  as  using  casein- 
agar),  but  the  principle  involved  is  the  same,  and  they  are  fully 
explained  and  discussed  in  the  article  by  Wiens.  Normal  blood  does 
not  contain  enough  leucocytes  to  cause  observable  cUgestion,  but  my- 
elogenous leukemia  blood  causes  distinct  chgcstion  while  lymphatic 
leukemia  does  not,  showing  that  it  is  the  polynuclears  and  myelocytes 
that  are  responsible.  Other  observations  fasten  the  proteolytic  activ- 
ity upon  the  neutrophile  granules.  Leucocytes  of  normal  human 
blood  will,  if  concentrated  enough,  cause  digestion  of  serum  plates, 
as  also,  of  course,  will  pus.  The  leucocytes  of  rabbits,  guinea  pigs, 
and  practically  all  animals  except  man,  apes  and  monkej^s,  are  de- 
void of  proteolytic  activity  demonstrable  by  the  plate  method.  Nor- 
mal serum,  both  homologous  and  heterologous,  exercises  a  strong 
inhibition  on  this  digestion,  so  that  it  is  necessary  to  have  an  excess  of 
leucocytes  present  to  obtain  the  reaction.  The  activity  of  leucocytic 
autolysis  is  indicated  by  the  observation  that  in  drawn  cerebrospinal 

12  Ito,  Jour.  Biol.  Chem.,  1916  (26),  173. 

'^  Full  bibliography  by  Wiens,  Ergebnisse  Phj-siol.,  1911  (15),  1;  Jochmann, 
Kolle  and  Wassermann's  Handbuch,   1912  (2),  1301. 


90  ENZYMES 

fluid  the  leucocytes  all  disappear  in  from  three  to  sixty-three  days,  and 
in  24  hours  the  count  has  been  observed  to  drop  from  392  to  6.^*  The 
leucocytic  enzymes  seem  to  be  very  resistant  against  chemicals, 
especially  against  formaldehyde,  so  that  museum  specimens  of  leuk- 
emic tissues  preserved  in  formalin  for  years  are  still  proteolytic.  Liver 
tissue  is  but  slightly  proteolytic  by  this  test,  spleen  more  so,  and  leuco- 
cyte-containing fluids,  such  as  saliva  and  colostrum,  are  quite  active. 
Pancreas  tissue,  has,  of  course,  strong  proteolytic  action,  but  it  is 
shown  to  be  distinct  from  the  leucocytic  protease  bj^  being  inhibited 
by  certain  sera  that  do  not  inhibit  the  leucocytic  protease.  In  gen- 
eral, tissues  do  not  cause  much  proteolysis  of  serum  plates  unless 
they  are  invaded  by  many  leucocytes,  which  applies  also  to  tumors, 
including  multiple  myelomas. 

Besides  proteases,  leucocytes  contain  other  enzymes.  ^^  To  quote 
the  summary  by  Morris  and  Boggs^^  "it  has  been  shown  that  the 
normal  and  pathological  neutrophile  leucocytes  and  myeloblasts 
contain  an  oxidase  and  probably  a  lipase  and  an  amylase;  myeloblasts 
contain  an  amylase.  In  lymphoid  tissues  two  proteases  and  a  lipase 
have  been  shown  to  exist.  In  leukemia  leukoprotease  has  been  demon- 
strated in  the  myeloid  variety  of  the  disease,  while  it  has  not  been 
found  in  chronic  lymphoid  leukemia.  Lipase  has  been  demonstrated 
in  two  cases  of  myeloid  leukemia,  and  oxidase  in  all  mj^-eloid  cases 
observed  in  which  the  neutrophilic  cells  were  present  in  excess." 
Jobling  and  Strouse,^^  confirming  Opie's  observation  of  two  distinct 
proteases  in  leucocytes,  find  also  evidence  of  an  ereptic  enzyme  acting 
in  either  acid  or  alkaline  fluids. ^^ 

Pneiunonia. — In  the  stage  of  resolution  lobar  pneumonia  presents 
a  striking  example  of  autolysis.  The  often-remarked  phenomenon 
that  the  lung  tissue  itself  is  not  in  the  least  affected,  while  the  dense 
contents  of  the  alveoli  are  rapidly  dissolved  and  removed  is  explained 
by  the  invariable  immunity  of  living  cells  to  digestive  enzymes.  Ex- 
cept for  some  slight  possible  assistance  by  the  alveolar  epithelium 
and  the  enzymes  of  the  serum,  the  enormous  and  rapid  digestion  of 
pneumonic  exudates  is  accomplished  by  the  leucocytic  enzymes.  The 
rapid  rate  of  digestion  may  be  accounted  for  by  the  absence  of  circu- 
lation within  the  alveolar  contents,  which  permits  the  leucocytes  to 

"  Bonaba,  Anales  de  le  Facultad  de  Med.,  1919  (4),  111. 

'^  According  to  Tschernoruzki  (Zeit.  physiol.  Chem.,  1911  (75),  216)  amylase, 
diastase,  catalase,  peroxidase,  and  nuclease,  but  not  lipase.  I  also  found  uricase 
absent  from  dog  leucocytes  (.Jour.  Biol.  Cnem.,  1909  (0),  321).  Fiessinger  and 
Marie  (Compt.  Rend.  Soc.  Biol.,  1909  (67),  177)  state  that  the  lymphocytes 
contain  lipase,  although  myeloid  cells  do  not.  (See  also  Resch,  Deut.  Arch.  klin. 
Med.,  191.5  (118),  179).  Leucocytes  are  also  said  to  contain  a  "lipoidase"  s])lit- 
ting  choline  from  lecithin  (Fiessinger  and  Clogne,  Compt.  Rend.  Acad.  Sci.,  1917 
(165),  730. 

i«  Arch.  Int.  Med.,  1911  (8),  806. 

"Jour.   Exj).   Med.,   1912  (16),  269. 

1*  Concerning  enzymes  of  normal  leucocytes  see  also  Fiessinger  and  Clogne, 
Ann.  de  M^-d.,  1917  (4),  445;  Parker  and  Franke,  Jour.  Med.  Res.,  1917  (37),  345. 


AUTOLYSIS  IN  PNEUMONIA  91 

act  unimpeded  by  the  anti-bodies  of  the  blood  plasma.  Digestion 
of  the  exudate  continues  after  death,  accounting  for  the  marked  dif- 
fuse softening  observed  in  pneumonic  lungs  in  bodies  kept  some  days 
before  autopsy.  As  long  ago  as  1888,  Kossel^^  mentioned  that  Fr. 
Miiller  had  found  that  glycerol  extracts  of  purulent  sputum  exhib- 
ited a  digestive  action  upon  fibrin  and  coagulated  protein,  whereas 
non-purulent  sputum  did  not  possess  this  property.  In  1877  Filehne 
extracted  ferments  in  the  same  way  from  the  sputum  in  gangrene 
of  the  lung;  Stolniknow,  in  1878,  found  a  similar  ferment  in  pneu- 
monic sputa,  and  Escherichin  1885  showed  that  the  proteolytic  action 
of  tuberculous  sputum  was  independent  of  putrefaction.  Other  early 
observations  of  similar  nature  are  reviewed  by  Simon, 2"  who  demon- 
strated the  presence  of  leucine  and  tyrosine  in  the  autolyzed  lungs. 
In  a  later  work  ^Miiller  reports  finding  three  grams  of  leucine  and 
tyrosine  in  a  pneumonic  lung,  as  well  as  lysine,  histidine,  and  purine 
bases  from  the  decomposed  nucleoproteins.  The  appearance  of  free 
purines  during  autolysis  of  pneumonic  lungs  has  been  investigated 
by  Mayeda,2i  Long  and  Wells. ^^  Boehm^^  isolated  histidine  and 
arginine  from  the  same  material.  Rietschel  and  Langstein^'*  found 
0.32  gm.  leucine  in  the  urine  of  a  pneumonic  child. 

Flexner^'  noted  that  autolysis,  while  very  rapid  in  the  gray  stage, 
is  but  slight  in  the  red  stage  (because  of  paucity  of  leucocytes)  and 
also  in  unresolved  pneumonia,  which  he  considers  as  due  to  some  inter- 
ference with  autolysis.  Silvestrini^^  found  that  in  gray  hepatization 
the  reaction  was  strongly  acid,  in  red  faintly  so;  the  graj"-  hepatization 
showed  more  peptone,  and  leucine  and  lactic  acid  were  both  demon- 
strable. A  fibrin-digesting  enzjmie  was  isolated,  and  milk  was  coagu- 
lated. 

Weiss^^  has  reported  finding  a  toxic  albumose  in  gray  pneumonic 
lungs.  Lord-*  has  found  in  pneumonic  lungs  a  proteolytic  enzyme 
active  at  pH  7.3  to  6.7,  but  inactive  at  higher  acidity;  also  an  enzjine 
sphtting  peptone  to  amino  acids  and  active  at  pH  8.0  to  4.8,  but 
most  active  at  6.3  to  5.2.  He,  therefore,  pictures  resolution  of  pneu- 
monic exudates  as  occurring  in  two  stages:  First,  proteolysis  while 
the  reaction  is  nearly  neutral,  and  later  as  the  acidity  increases  the 
cleavage  of  the  peptone  increases.  He  also  finds  that  the  pneumo- 
cocci  cannot  long  survive  a  reaction  more  acid  than  pH  6.8,  and 
their  dissolution  takes  place  at  reaction  from  6.0  to  5.0,  which  is  of 

19  Zeit.  f.  klin.  Med.,  1888  (13),  149. 

20  Deut.  Arch.  klin.  Med.,  1901  (70),  604. 
"1  Deut.  Arch.  klin.  Med.,  1910  (98),  587. 
22 /Wd,    1914   (115),   377. 

23  Ibid.,  1910  (98),  583. 

24  Biochem.  Zeit.,  1906  (1\  75. 

25  Univ.  of  Penn.  Med.  Bull.,  1903  (16),  185. 

-^  Bull.  del.  Soc.  Eustachiana,  1903,  abst.  in  Biochem.  Centralbl.,  1903  (1),  713. 
"  Arch.  Int.  Med.,  1919  (23),  395. 
28  Jour.  ExD.  Med.,  1919  (30),  379. 


92  ENZYMES 

significance  in  view  of  the  observation  that  pneumonic  lungs  are  more 
acid  than  normal  organs,  acidity  as  high  as  pH  6.0  to  5.4  having  been 
found. 2^ 

Rzentkowski^"  found  an  increase  of  non-coagulable  nitrogen  in  the 
blood  of  pneumonics,  probably  resulting  from  autolysis  in  the  exu- 
date. According  to  Dick^^  the  blood  serum  after  the  crisis  contains 
an  enzyme  which  acts  specifically  on  the  pneumococcus  proteins. 
Petersen  and  Short^^  found  an  increase  of  serum  ereptase  in  the  blood 
preceding  or  accompanying  crisis  or  lysis,  and  suggest  that  it  may  have 
a  function  in  attacking  the  toxic  protein  fragments.  In  the  liver  during 
experimental  pneumococcus  septicemia,  autolysis  is  said  to  be  increased 
in  rate..^^  Almagia**  suggests  that  the  bactericidal  action  of  the  prod- 
ucts of  fibrinolysis  in  pneumonia  may  be  of  importance  in  checking 
the  disease. 

Necrotic  Areas. — Jacoby^^  found  that  if  a  portion  of  a  dog's  liver 
was  ligated  oF  and  the  animal  kept  alive  for  some  time,  the  necrotic 
tissue  contained  the  same  products  that  he  had  obtained  in  experi- 
mental autolysis.  The  absorption  of  necrotic  tissues  generallj^  is 
ascribable  to  either  autolysis  or  heterolysis.  Presumably  there  is  no 
great  difference  in  the  self-digestion  of  an  organ  which  is  necrotic 
because  its  blood  supply  is  cut  ofT,  and  of  a  similar  organ  removed 
from  the  body  aseptically  and  allowed  to  undergo  aseptic  autolysis 
in  an  incubator.  At  the  periphery  there  might  be  some  effects  pro- 
duced in  vivo  by  the  inhibitive  action  of  the  serum  or  the  digestive 
action  of  the  leucocytes,  but  beyond  that  no  marked  differences  are 
to  be  expected.  In  both  cases  asphyxia  is  present,  leading  to  increased 
acidity,  without  which  little  if  any  autolysis  can  occur.  It  has  been 
found  that  in  experimental  infarction  of  the  kidney  there  develops 
sufficient  acidity  to  permit  of  autolysis,  and  the  hydrolysis  of  the 
proteins  increases  with  the  development  of  acidity  (Straus  and  Morse.^®) 

A  study  of  the  relation  of  autolysis  to  the  histological  changes  that 
occur  in  necrotic  areas  by  Wells^'^  gave  evidence  that  there  occurs 
early  a  decomposition  .of  the  nucleoproteins  of  the  nuclei,  which  is 
probably  brought  about  by  the  intracellular  autolytic  enzymes.  The 
liberation  of  the  nucleic  acid  and  the  reduction  in  the  bulk  of  nuclear 
material  through  the  digestion  away  of  the  protein  is  probably  the 
cause  of  the  pycnosis  observed  in  necrotic  areas.  Later  the  nucleic 
acids  are  further  decomposed  through  the  special  enzymes  described 
by  Jones,  Sachs,  and  others,  the  "nucleases."     This  is  prosuniably 

-3  Jour.  Amer.  Med.  Assoc,  1919  (72),  1364. 

30  Virchovv's  Arch.,  190.5  (179),  405. 

31  Jour.  Infect   Dis.,  1912  (10),  383. 
'2  Jour.  Infect.  Dis.,  1918  (22),  147. 

"  Medigrcceanu.  Jour.  Exp.  Med.,  1914  (19),  31. 
3^  Festschr.  for  Celli,  Torino,  1913,  p.  459. 
^oZcit.  physiol.  Chein.,  1900  (30),  149. 
'«  Proc.  Soc.  Exp.  Biol.  Med.,  1917  (14).  171. 
"  Jour.  Med.  Research,  1900  (15),  149. 


AUTOLYSIS  IN  NECROTIC  TISSUES  93 

the  cause  of  the  h).ss  of  nueleai-  staining  so  characteristic  of  necrosis. 
That  these  changes  are  due  to  the  intracellular  enzymes  was  shown  by 
implanting  in  animals  pieces  of  sterile  tissues,  the  enzymes  of  which 
had  been  destroyed  by  heating;  these  were  found  to  undergo  altera- 
tions only  after  several  weeks,  and  then  as  the  result  of  the  action 
upon  them  of  invading  leucocytes.  The  slow  rate  of  autolysis  that 
occurs  in  infarcts  and  other  aseptic  areas  is  presumably  due  in  part  to 
the  action  of  the  antibodies  of  the  serum,  for  it  was  found,  experimen- 
tally, that  the  histological  changes  of  autolysis  when  the  tissues  are 
placed  in  heated  serum  proceed  about  twice  as  rapidly  as  when  they 
are  placed  in  fresh  serum.  Chemotactic  substances  do  not  seem  to 
be  formed  in  aseptic  dead  tissues,  but  the  slow  absorption  of  such 
tissues  is,  however,  finalh^  accomplished  by  the  leucocytes  acting 
from  the  periphery,  there  being  little  actual  autolysis  of  the  dead 
cells  b}'  their  own  enzj'mes.  The  rapidit}'  with  which  autolytic 
changes  occur  in  different  organs,  as  indicated  by  the  disappearance 
of  nuclear  staining,  seems  to  be  about  as  follows:  (1)  Liver,  kidney 
(epithelium  of  convoluted  tubules);  (2)  spleen,  pancreas;  (3)  kidney 
(collecting  tubules,  straight  tubules,  glomerules);  (4)  lung  (alveolar 
and  bronchial  epithelium);  (5)  thyroid;  (6)  myocardium;  (7)  volun- 
tary muscle;  (8)  skin  (epithelium);  (9)  brain  (cortical  cells).  Stroma 
cells  seem  to  be  attacked  chiefly  by  enzymes  from  the  parenchyma 
cells.  Of  all  cellular  elements,  the  endothelium  of  the  vessels  seems 
to  have  the  greatest  resistance  to  both  autolysis  and  heterolysis. 

The  finer  structural  changes  of  aseptic  autolysis  of  liver  in  salt  so- 
lution, have  been  carefuly  studied  by  Launoy,^^  who  notes  a  period 
of  relative  latency  (20  to  24  hours  at  38°) ,  followed  by  rapid  changes 
in  both  cytoplasm  and  nucleus,  associated  with  the  appearance  of 
myelin  forms.  Dyson^^  describes  loss  of  the  Altmann's  granules  in 
autolyzing  cells.  Cruickshank*"  states  that  when  aseptic  autolysis 
of  tissues  kept  in  a  moist  chamber  is  observed  microscopically  the 
changes  are  slower,  and  there  is  less  solution  of  the  cytoplasm,  but  in 
general  the  results  are  much  the  same.  No.  fat  could  be  found  by 
special  stains.  Fetuses  that  have  undergone  aseptic  autolysis  in  the 
uterus  show  complete  loss  of  nuclei  in  5  to  6  days,  a  stage  correspond- 
ing to  8  to  15  days  autolysis  in  the  moist  chamber.  In  experimental 
nephritis  Simons^^  observed  a  decreased  autolysis  of  the  kidneys. 

Degenerated  nervous  tissue  also  undergoes  a  slow  autolj^sis  which, 
according  to  Noll,*^  results  in  the  splitting  of  "protagon"  with  hbera- 
tion  of  lecithin.     ]\Iott,  Halliburton, ■'•^  Donath,  and  others  have  shown 

'*  Ann.  Inst.  Pasteur,  1909  (23),  1. 

"9  Jour.  Path,  and  Bact.,  1912  (17),  12;  also  Aschoff,  Verb.  deut.  Path.  Gesellsch, 
1914  (17),  109. 

"  Jour.  Path,  and  Bact.,  1911  (16),  167. 

"Biochem.  Zeit.,  1S14  (67),  483. 

"  Zeit.  physiol.  Cheiu.,  1899  (27),  390. 

*^  General  resume  in  Ergebnisse  der  Physiol.,  1904  (4),  24. 


94  ENZYMES 

that  in  nerve  destruction  lecithin  is  spht  up  with-hberation  of  cho- 
line (see  "Choline")-  Koch  and  Goodson^*  found  that  degenerated 
nervous  tissue  is  characterized,  chemically,  by  containing  a  relatively 
increased  amount  of  nucleo-proteins,  with  an  absolute  decrease  in 
solid  constituents,  while  the  lecithins  are  greatly  altered. 

In  caseation  autolysis  is  very  slight,  as  is  shown  by  the  persistence 
of  the  caseous  material  for  long  periods  of  time  without  absorption. 
Presumably  the  toxin  of  tuberculosis  destroys  the  autolytic  ferments 
of  the  cells  it  kills, ^^  and  as  there  is  little  chemotactic  influence,  leuco- 
cytes do  not  enter  the  caseous  area.  Jobling  and  Petersen^^  find 
evidence  that  the  soaps  of  unsaturated  fatty  acids  present  in  tubercles 
are  responsible  for  the  inhibition  of  digestion.  Spiethoff^''  found 
that  pure  caseous  material  is  usually  free  from  even  traces  of  albumose 
and  peptone,  but  the  caseous  material  at  the  periphery  mixed  with 
tissue  elements  contains  them  in  very  small  quantities,  suggesting  that 
at  the  periphery  of  caseous  areas  some  slight  autolysis  does  occur.  The 
fact  that  B.  tuberculosis  is,  itself,  very  poor  in  proteolytic  enzymes  as 
compared  with  most  other  bacteria  may  be  another  factor.  When 
leucocytes  are  attracted  into  a  tuberculous  focus  softening  goes  on 
rapidly,  showing  that  there  is  no  loss  of  digestibility  of  the  caseous 
material,  but  merely  a  lack  of  enzymes.  Pus  from  a  cold  tuberculous 
abscess  will  not  digest  fibrin,  but  if  iodoform  is  injected,  leucocytes 
enter  in  great  numbers,  softening  is  rapid,  and  the  pus  will  then  di- 
gest fibrin  (Heile^^).  On  serum  plates  tuberculous  pus  produces  no 
digestion  unless  a  secondary  infection  or  other  cause  has  resulted  in 
a  local  accumulation  of  leucocytes. ^^  Tuberculous  material  contains, 
like  the  lymphocytes,  an  enzyme  which  is  proteolytic  in  acid  media 
and  which  is  inhibited  by  normal  serum  (Opie  and  Barker'*^). 

Correlation  of  Histological  and  Chemical  Changes. — A  careful  study  of  the 
relationship  of  the  chemical  chan}.';es  produced  by  autolysis,  to  the  histological 
changes  of  necrosis  and  autolysis,  has  been  made  by  H.  J.  Corper,^"  and  colored 
plates  published  together  with  analytical  figures  make  it  possible  to  correlate  at 
a  glance  the  structural  and  chemical  changes  of  necrobiosis.  Corper  found  that 
in  the  early  stages,  characterized  by  a  high  grade  of  pycnosis  but  no  further  nu- 
clear changes,  the  nucleins  are  still  intact;  but  with  well  developed  karyorrhexis 
and  beginning  karyolysis,  some  ten  per  cent,  of  the  nuclein  nitrogen  has  become 
soluble  in  the  form  of  purine  bases.  When  karyolysis  is  completed  so  that  no  more 
nuclei  remain  in  a  stainable  condition,  only  twenty-eight  ])er  cent,  of  the  nucleo- 
proteins  was  found  to  have  been  dccom])oseil  to  free  purine  bases,^'  the  remaining 
seventy-two  per  cent,  being  intact  although  unstainable.  This  rather  surpris- 
ing observation  indicates  that  the  stainable  chromatin  rejjresents  but  about  one- 
fourth  of  the  nucleins  of  the  cell,  which  is  in  accord  with  the  views  of  Hamnuirsten 

«Amer.   .Jour.   Physiol.,    1906   (15),   272. 

"  However,  Pesci  (Pathologica,  1912  (3),  144)  states  that  tuberculin  increases 
autolysis  in  vitro. 

"Uour.  Exp.  Med.,  1914  (19),  383. 

■"  Cent.  f.  inn.  Med.,  1904  (25),  481. 

"8  Zeit.  klin.  Med.,  1904  (55),  508. 

"Jour.  Exper.  Med.,  1909  (11),  686. 

^"Jour.    Exper.    Med.,    1912   (15),   429. 

"  Marshall  (Jour.  Biol.  Chem.,  1913  (15),  81)  has  also  found  that  much  of 
the  nucleic  acid  remains  unaltered  in  autolysis  of  thymus. 


AUTOLYSIS  OF  THE  LIVER  95 

and  others.  The  lecithin  disintegrates  somewhat  more  completely,  about  one- 
half  or  two-thirds  beinp  disintegrated  by  the  time  nuclear  destruction  is  complete, 
after  whicli  this  and  all  other  autolytic  change  is  slow.  The  change  from  coagul- 
able  to  non-coagulable  forms  of  nitrogen  was  as  follows:  Normal  spleen,  non- 
coagulable  nitrogen,  5.7  per  cent,  of  the  total;  stage  of  marked  pycnosis,  without 
rhexis  or  lysis,  7.4  per  cent.;  stage  of  karyonhexis  and  early  karyolysis,  2(j.5  jjer 
cent. ;  stage  of  complete  karyolysis,  ;^0.3  per  cent.  That  is,  when  nuclear  structures 
in  the  spleen  have  lost  their  staining  properties  entirely  through  autolysis,  about 
72  per  cent,  of  the  nuclein  nitrogen,  50  per  cent,  of  the  insoluble  phosphorus  com- 
pounds. 70  per  cent,  of  the  coagulable  nitrogen,  and  about  two-thirds  of  the 
lecithin  are  still  intact. 

Liver  Degenerations. — ^Thc  relation  of  the  disintegration  observed 
in  phosphorus-poisoning  and  acute  yellow  atrophy  to  the  experimental 
autolysis  of  the  liver  has  been  the  object  of  much  study.  Salkowski 
originally  pointed  out  that  the  same  products  were  found  in  the  blood, 
urine,  and  liver  tissue  in  acute  yellow  atrophy  as  are  produced  in 
autolysis.  Jacoby^^  found  that  the  livers  of  dogs,  taken  just  as  the 
animals  were  dying  of  phosphorus-poisoning,  contained  free  leucine 
and  tyrosine;  also,  he  found  that  the  rate  of  autolysis  of  such  livers 
after  removal  from  the  body  was  much  greater  than  in  normal  livers. 
The  oxidizing  ferments  (aldehydase)  are  not  destroyed  by  the  proc- 
ess. He  found  that  addition  of  minute  amounts  of  phosphorus  to 
liver  enzymes  did  not  increase  their  proteolytic  power;  nevertheless, 
he  seems  inclined  to  assume  that  in  phosphorus-poisoning  alteration 
in  the  autolytic  enzj^mes  is  an  important  factor  in  the  liver  degen- 
eration. It  would  seem  much  more  probable  that  phosphorus  is  a 
poison  that  kills  cells  and  does  not  destroy  their  autolytic  enzymes, 
hence  favoring  autolysis.  The  liver  degeneration  following  chloro- 
form poisoning  may,  perhaps,  be  explained  in  a  similar  way,  the  cells 
behaving  exactly  as  bacteria  would  do  under  the  same  conditions. 
Taylor^^  has  analyzed  several  livers  in  degenerative  conditions  for 
amino-acids  and  found  them  only  in  one  liver,  which  showed  necrosis 
probabl}^  due  to  chloroform  poisoning,  and  which  was  from  a  case 
clinically  resembling  acute  yellow  atrophy.  Here  he  obtained  4  gm. 
of  leucine,  2.2  gm.  of  tyrosine,  and  2.3  gm.  of  arginine  nitrate.  Wald- 
vogel  and  Tintemann,^*  in  phosphorus  livers,  found  an  increase  in 
protagon,  jecorin,  fatty  acids,  cholesterol,  and  neutral  fat,  while 
lecithin  w^as  decreased.  Wakeman"  found  arginine,  histidine,  and  ly- 
sine decreased  in  phosphorus  livers  in  proportion  to  the  total  nitro- 
gen, indicating  that  the  protein-splitting  enzyme  in  this  condition 
either  picks  out  certain  varieties  of  proteins  first,  or  removes  the 
nitrogen-rich  constituents  most  rapidly. ^^ 

52  Zeit.  f.  physiol.  Chem.,  1900  (30),  174. 

"  Univ.  of  Calif.  Public,  (pathol.),  1904  (1),  43. 

"  Cent.  f.  Path.,  1904  (15),  97. 

"  Berl.  klin.  Woch.,  1904  (41),  1067. 

5^  Considerable  quantities  of  amino-acids  of  various  sorts  have  been  isolated 
from  the  liver  in  acute  vellow  atrophv  and  chloroform  necrosis  bv  Wells  (Jour. 
Exper.  Med.,  1907  (9),  627;  Jour.  Biol.  Chem.,  190S  (5),  1-29);  but  the  value  of 
these  figures  is  questionable  because  it  is  possible  that  the  alcohol  in  which  the 
tissues  were  kept  before  analysis  was  not  strong  enough  entirely  to  prevent  au- 
tolysis (Wells  and  Caldwell.  Jour.  Biol.  Chem.,  1914  (19),  57). 


96  ENZYMES      " 

It  is  probable  that  many  poisons  may  injure  the  liver  cells  to  such 
an  extent  that  they  cannot  maintain  their  normal  chemical  equili- 
brium, but  without  destroying  the  autolytic  enzymes.  When  this 
occurs,  the  liver  undergoes  autolysis,  and  we  get  marked  degenerative 
changes  with  appearance  of  amino-acids  in  the  blood  and  urine, 
reduction  in  coagulability  of  the  blood  and  numerous  hemorrhages, 
giving  a  picture  both  clinically  and  anatomically  more  or  less  like  that 
of  typical  acute  yellow  atrophy.  Chloroform  is  a  poison  that  stops 
cell  activities  without  destroying  the  proteolytic  enzymes,  hence 
the  cells  undergo  autolysis,  and,  as  a  result,  we  have  many  cases  of 
what  appears  to  be  acute  yellow  atrophy  following  chloroform  anes- 
thesia. The  liberation  of  HCl  in  the  liver  cells  during  chloroform 
poisoning,  as  demonstrated  by  Evarts  Graham, ^'^  may  be  largely  re- 
sponsible for  the  rapid  disintegration  of  the  liver  in  this  condition. ^^ 
(See  ''Acute  Yellow  Atrophy, "  Chap,  xx.)  Probably  the  liver  changes 
in  puerperal  eclampsia,  and  in  streptococcus  and  other  septicemias  are 
of  a  similar  nature. ^^  Autolysis  of  fatty  livers  in  tuberculosis  is  said 
to  yield  more  lactic  acid  than  the  livers  from  other  conditions  (Yous- 
souf).60 

Postmortem  changes  are  undoubtedly  due  to  two  factors,  bac- 
terial action  and  autolysis.  In  tissues  kept  at  a  low  enough  tempera- 
ture to  exclude  bacterial  action,  but  not  so  low  as  absolutely  to  stop 
enzyme  action, ^^  there  occurs  a  slow  autolysis;  this  constitutes  the 
"ripening"  process  of  meat.  Fish  flesh  may  also  ripen  when  made 
sterile  in  saturated  salt  solutions,  as  Schmidt-Nielsen^"^  has  shown 
occurs  with  salted  herrings,  oxy-acids  and  xanthine  bases  being  promi- 
nent among  the  products.  The  softening  of  muscles  in  rigor  mortis  is 
probably  also  an  autolytic  manifestation,  as  muscles  contain  proteases 
acting  best  in  acid  medium,  and  the  muscle  is  known  to  become  in- 
creasingly acid  after  circulation  ceases  within  it.  The  short  duration 
of  rigor  mortis  when  the  body  is  kept  warm,  and  its  early  disappear- 
ance when  death  has  been  preceded  by  muscular  exhaustion  (which 
increases  the  acidity),  agree  with  this  view.  The  oarly  postmortem 
softening  of  many  organs  in  pathological  conditions  is  also  probably 
an  autolytic  manifestation.  Flexner-'^  has  called  attention  to  this 
in  relation  to  the  softening  of  the  parenchymatous  organs  in  acute 
infectious  diseases,  such  as  typhoid  and  septicemia.  Scluunm  noted 
great  autolytic  activity  in  a  swollen  spleen  from  a  case  of  perityphlitis. 

"  Jour.  Exp.  Med.,  1915  (22),  48. 

68  Quinan  (.Tour.  Med.  Res.  1915  (.32),  73)  found  no  ehanfre  in  the  rate  of 
in  tritro  autolysis  of  liver  tissue  from  experimental  cliloroform  poisoning.  It  was 
found  increased  by  phlorhizin  (Satta  and  Fasiani,  Arch.  di.  Fisiol.,  1913  (11), 
391). 

f-a  Wells,  Jour.  Amer.  Med.  Assoc,  190G  (40),  341. 

6"  Virchow's  Arch.,  1912  (207),  374. 

*'  Some  chemical  chaufre  may  taivc  i)lac.e  at  temiieratures  as  low  as  —2  and  —  14 
(Costantino,  Arch.  farm,  sper.,  1917  (24),  255). 

«Mrofmeister's  Heitra^rc,  l«K)3  (3).  2(57. 


AUTOLYSIS  IN  INFECTION  97 

Histological  changes  are  produced  by  autolysis  in  the  organs  after 
death  that  are,  as  might  be  expected,  much  like  those  seen  in  necrotic 
areas. ^^  At  first  the  changes  resemble  those  of  parenchymatous  de- 
generation (cloudy  swelling),  and  often  there  is  an  apparent  increase 
in  fat,  which  is  probabl}'  due  to  liberation  of  masked  fat  through  the 
destruction  of  the  protein.*''  Nuclear  staining  is  lost  (karyolysis) , 
and  eventually  even  cell  forms  become  indistinguishable.  (See  p.  94 
on  structural  changes  of  postmortem  autolysis.) 

•  Still-born  children  that  have  been  carried  for  some  time  after  death 
usually  show  considerable  disintegration  of  the  viscera,  especially  the 
liver.  This  is  undoubtedly  due  to  autolysis,  which  Schlesinger^^ 
has  shown  can  begin  before  birth  if  the  fetus  dies  in  utero. 

Autolysis  in  Relation  to  Infection. — According  to  Conradi^Hhe 
substances  produced  in  tissue  autolysis  have  a  decided  inhibiting  effect 
upon  bacteria,  which  apparently  depends  upon  the  antiseptic  proper- 
ties of  the  aromatic  derivatives  that  are  split  out  of  the  protein  mole- 
cule in  autolysis.  This  action  is  manifested  not  only  in  vitro,  but  the 
autolytic  products  will  also  render  harmless  lethal  doses  of  certain 
bacteria  if  they  are  injected  simultaneously  with  the  bacteria  into  an 
animal.  One  specific  class  of  products  of  autolysis  which  is  strongly 
bactericidal  is  the  soaps. ^^  It  may  well  be  questioned,  however, 
whether  enough  of  these  substances  ever  accumulates  in  infected 
tissues  during  intra  vitam  autolysis  to  have  much  affect  upon  the  in- 
fecting bacteria;  yet  this  property  may  possibly  explain  the  steriliza- 
tion of  old  pus  collections  and  similar  infected  localized  accumulations 
within  the  body.  The  bacteria  themselves  also  produce  autolytic 
products  that  are  pow^erfully  bactericidal.     (See "Bacteria,  "Chap. iv.) 

Blum®^  says  that  the  autolytic  products  of  Ij^mph-glands  neutra- 
lize tetanus  toxin,  but  are  inactive  against  diphtheria  toxin  and  cobra 
venom.  Products  from  other  autolyzed  organs  and  from  fresh  lymph- 
glands  were  without  influence  on  the  tetanus  toxin.  The  antitoxic 
principles  of  the  autolytic  product  were  destroyed  by  heating,  weak- 
ened by  acids  and  alkalies,  and  in  other  respects  showed  prop- 
erties strikingly  like  those  of  true  antitoxin.  It  is  quite  possible  that 
bacterial  toxins  may  be  destroyed  by  autolytic  enzymes,  for  Baldwin 
and  Levene^^  have  shown  that  trypsin,  pepsin,  and  papain  destroy 
tetanus  and  diphtheria  toxin,  while  tuberculin  is  destroyed  by  trypsin, 
but  not  readily  by  pepsin,  possibly  because  it  is  of  a  nucleoprotein 

«^  More  fully  discussed  bj'  Wells,  Jour.  Med.  Research,  1906  (15),  149. 

^*  Siegert  (Hofmeister's  Beitr.,  1901  (1),  114)  found  no  actual  increase  in  fats 
and  fatty  acids  in  autolysis  even  when  an  increase  was  apparent  histologically , 
although  ether-soluble  materials  of  other  nature  than  fat  mav  be  increased.  See 
also  Hess  and  Saxl,  Virchow's  Arch.,  1910  (202),  149. 

"  Hofmeister's  Beitr.,  1903  (4),  87. 

««  Hofmeister's  Beitr.,  1901  (1),  193.     See  also  Bilancioni"  and  Almagia.^* 

8'  See  Lamar,  Jour.  Exp.  Med.,  1911  (13),  1. 

88  Hofmeister's  Beitr.,  1904  (5),  142. 

"  Jour.  Med.  Research,  1901  (6),  120.  ^ 

7 


98  ENZYMES 

nature.  The  leucocytic  proteases,  however,  seem  not  to  attack  either 
toxins  or  Uving  bacteria  (Jochmann).  Bertohni'°  states  that  auto- 
lyzing  hver  will  destroy  diphtheria  toxin. 

On  the  other  hand,  there  are  many  pathogenic  bacteria  which  do 
not  secrete  their  toxic  materials,  but  store  them  up  within  the  cell 
body,  e.  g.,  typhoid,  cholera,  and,  indeed,  the  majority  of  pathogenic 
forms.  These  endotoxins  are  probably  hberated  from  the  bacteria 
only  through  digestion  of  their  cells,  either  by  their  own  autolytic 
enzymes  or  by  the  enzymes  of  the  infected  tissues  and  leucocytes. 

Leukemia. — The  abundant  elimination  of  uric  acid  and  other  pu- 
rine bodies  in  the  urine  in  leukemia  testifies  to  the  great  amount  of 
destruction  of  nucleoprotein  that  is  going  on  during  the  disease,  and 
these  are  probably  derived  from  the  autolysis  of  leucocytes,  which  per- 
haps depends  on  the  relatively  large  proportion  of  leucocytes  to 
serum.  Schumm'^^  has  found  that  leukemic  spleens  and  bone  marrow 
autolyze  rapidly  and  completely,  and  he  isolated  many  of  the  cleavage 
products  of  protein  digestion  from  such  autolysates. 

Leucocytes  from  myeloid  leukemia  hquefy  alkaline  gelatin  vigor- 
ously, but  those  from  lymphatic  leukemia  do  not;  the  liquefaction  is 
inhibited  by  normal  serum  (Stern  and  Eppenstein).''^  By  the  serum 
plate  method  this  observation  has  been  much  extended,  and  the  hetero- 
lytic  action  of  the  leucocytes  has  been  found  limited  to  the  neutro- 
phile  granules.  In  neutral  media  evidence  is  obtained  of  the  presence 
of  protease  in  the  lymphocytes  of  chronic  lymphatic  leukemia  and  the 
leucocytes  of  acute  and  chronic  myeloid  leukemia;  maltase,  lipase  and 
amylase  are  found  in  both  types  of  cells,  and  oxidase  in  the  granular 
cells  derived  from  the  marrow  (Morris  and  Boggs).'^^  v.  Jaksch,"^ 
Erben,'^^  and  others  have  noted  the  occurrence  of  peptones  and  albu- 
moses  in  leukemic  blood,  particularly  if  removed  postmortem.  The 
improvement  in  leukemia  that  follows  .r-ray  treatment  is  associated 
with  an  increased  nitrogen  elimination,  probably  due  to  autolysis  of 
disintegrating  cells, '^^  although  a;-rays  have  no  appreciable  effect  upon 
the  leucocytic  proteases  in  vitro  (Miiller  and  Jochmann).  (See  also 
"Leukemia,"  Chap,  xiii.) 

Tumors. — Probably  because  of  the  great  amount  of  necrosis  that 
is  constantly  going  on  in  all  malignant  growths,  with  subsequent  di- 
gestion of  the  dead  cells,  autolytic  products  are  present  in  them  in 
very  considerable  amounts.     This  was  first  demonstrated  by  Petry," 

'"'  Biochem.  Zeit.,  1913  (48),  448. 

^'  Hofmeister's  Beitr.,  1903  (3),  576;  1905  (7),  175. 

^^  See  discussion  of  leucocytic  enzymes,  p.  89.  Longcope  and  Donhauser  (Jour. 
Exper.  Med.,  1908  (10),  G18)  found  proteases  in  the  large  lymphocytes  in  acute 
leukemia,  which  were  most  active  in  an  alkaline  medium. 

"  Arch.  Int.  Med.,  1911  (8),  806. 

'^  Zeit.  f.  physiol.  Chem.,  1892  (16),  243. 

'^Zeit.  f.  klin.  Med.,  1900  (40),  282;  Zeit.  f.  Heilkunde,  1903  (24),  70;  Ilof- 
meiter's  Beitr.,  1904  (5),  461. 

'6  Musser  and  Edsall,  Univ.  Penn.  Med.  Bull,  1905  (18),  174. 

"  Zeit.  f.  physiol.  Chem.,  1899.(27),  398;  Hofmeister's  Beitr.,  1902  (2),  94. 


AUTOLYSIS  IN  TUMORS  99 

who  fouiul  that  cai-ciiionias  of  the  breast  contained  much  of  their 
nitrogen  in  compounds  not  coagulated  by  heat,  while  in  the  normal 
gland  practically  all  is  coagulable.  He  also  demonstrated  an  autolytic 
property  in  tumor  tissue,  showing  that  tumor  cells  do  not  difTcr  in  this 
respect  from  normal  cells.  Beebe'**  found  products  of  autolysis  con- 
stantly present  in  several  tumors;  namely,  a  carcinoma  of  the  broad 
ligament,  a  hypernephroma,  an  angiosarcoma,  and  a  round-cell 
sarcoma. 

Ncuberg^'-^  found  that  while,  according  to  other  observers,  most 
enzymes,  as  well  as  bacteria,  are  very  susceptible  to  the  action  of 
radium  rays,  the  autolytic  enzymes  of  cancer  cells  are  an  exception, 
for  cancer  tissue  exposed  to  radium  undergoes  autolysis  much  faster 
than  cancer  tissue  not  exposed  to  radium;  x-rays  are  less  active  in 
this  respect.  He  attributes  the  effects  of  radium  on  cancer  to  its 
deleterious  effects  on  the  oxidizing  and  other  enzymes  of  the  cells, 
destroying  their  activities,  which  results  in  destruction  of  the  cells 
by  the  autolytic  enzymes.^"  A  cancer  of  the  stomach  was  found  to 
contain  autolytic  enzymes  capable  of  digesting  lung  tissue  (pepsin 
was  excluded)  and  autolyzed  cancers  yielded  much  pentose.  Blu- 
raenthal  and  Wolf  ^^  believe  that  tumor  tissues  have  particularly  active 
autolytic  enzymes,  since  liver  tissue  added  to  tumor  tissue  underwent 
autolysis  much  more  rapidly  than  normal;  but  tumors  do  not  cause 
digestion  of  serum  plates  unless  many  leucocytes  are  present  (Mliller 
and  Kolaczek).^-  Cancer  extracts  digest  peptids  in  ways  cUfferent 
from  normal  tissues,  which  seems  to  indicate  some  fundamental  ab- 
normality in  their  metabolism  (Abderhalden,^^  Neuberg).^^  The  al- 
most constant  presence  in  gastric  juice  of  patients  with  carcinoma 
of  the  stomach,  of  ereptases  hydrolyzing  proteoses  and  peptids,  is 
generally  attributed  to  the  disintegration  of  the  cancer  with  libera- 
tion of  these  enzymes. ^^  Tumors  also  contain  nuclease^Ho  disintegrate 
their  nucleic  acid,  and  the  same  outfit  of  purine-splitting  enzymes 
as  normal  tissues,"  so  that  in  regard  to  the  nucleoproteins  of  tumors, - 
autolysis  follows  the  same  course  as  in  normal  tissues. 

'8  Amer.  Jour.  Physiol.,  1904  (11),  139. 

^^Zeit.  f.  Krebsforschung,  1904  (2),  171;  Berl.  klin.  Woch.,  190-4  (41),  1081; 
ibid.,  1905  (42),  118;  Arb.  Path.  Inst.  Berlin,  1906,  p.  593. 

8«Wohlgeinuth,  Berl.  klin.  Woch.,  1904  (41),  704,  found  that  autolysis  in 
tuberculous  lung  tissue  was  three  or  four  times  more  rapid  when  exposed  to 
radium  rays.  ^Heile  (Arch.  klin.  Chir.,  1905  (77),  107)  looks  upon  the  favorable 
effects  of  x-rays  as  partly  produced  by  their  liberation  of  autolytic  enzymes  from 
the  leucocytes. 

«'  Med.  Klinik.',  1905  (1),  No.  7. 

^■-  Miiller  and  Kolaczek,  Miinch.  med.  Woch.,  1907  (54),  354;  Hess  and  Saxl 
}\ifn.  khn.  Woch.,  1908  (21),  1183;  Kepinow,  Zeit.  f.  Krebsforsch.,  1909  (7)' 
517.  ' 

"Zeit.  physiol.  Chem.,  1910  (66),  277. 

8*  Biochem.  Zeit.,  1910  (26),  344. 

soggp  Jacques  and  Woodyatt,  Arch.  Int.  Med.,  1912  (10),  5G0;  Hambureer 
Jour.  Amer.  Med.  Assoc,  1912  (59),  847.  ' 

^^  Goodman,  Jour.  Exp.  Med.,  1912  (15),  477. 

"  Wells  and  Long,  Zeit.  Krebforsch.,  1913  (12),  598. 


100  ENZYMES 

The  non-cancerous  livers  of  cancerous  patients  were  found  by  Yous- 
souf^°  to  produce  more  lactic  acid  during  antiseptic  autolysis  than 
did  livers  in  other  conditions.  Autolysis  of  organs  of  cancer  patients 
is  about  as  rapid  as  normal  (ColwelP^).  Several  observations  have 
suggested  that  tumor  tissues  might  contain  proteolytic  enzymes  dif- 
fering from  those  of  normal  tissues  especially  in  their  ability  to  digest 
heterologous  normal  tissues,  but  at  present  this  work  needs  confirma- 
tion and  amplification  before  it  can  carry  the  weight  of  speculation 
which  has  been  heaped  upon  it.^^ 

Micheli  and  Donati^°  attribute  the  hemolytic  properties  possessed 
by  extracts  of  malignant  tumors  to  the  products  of  autolysis  that  are 
present,  which  Petry  has  also  demonstrated  to  produce  hemolj'sis. 
Emerson^i  attributes  the  disappearance  of  HCl  from  the  gastric  juice 
in  carcinoma  of  the  stomach  to  neutralization  by  basic  products 
of  autolysis,  a  hypothesis  that  may  well  be  questioned.  (See  also 
"Tumors,"  Chap,  xix.) 

Various  other  intracellular  enzymes  have  been  described,  which  for  the  most 
part  have  as  yet  no  significance  in  pathology.  An  exception  is  fibrin  ferment, 
■which  will  be  considered  fully  in  discussing  thrombosis.  Ferments  coagulating 
milk  seem  'to  be  widely  spread  in  the  tissues.  The  precipitation  of  plastein  from 
proteose  solution  by  organ  extracts  (Niirnberg)  may  be  either  the  effect  of  a 
•coagulating  ferment  or  due  to  reverse  action  of  the  proteases.  Ferments  split- 
ting specifically  maltose,  lactose,  sucrose,  glucosides,  and  nucleoproteins  have 
been  described,  and  the  glycogenolytic  ferment  is  probably  nearly  imiversally  pres- 
ent. Other  enzymes  decomposing  amino-acids  into  ammonium  compounds  may 
also  exist.  The  enzymes  acting  specifically  upon  the  nucleic  acids  and  the  purine 
bodies  are  discussed  in  Chapter  xxiii. 

88  Arch.  Middlesex  Hosp.,  1910  (19),  55. 

83  See,  for  example,  Rulf,  Zeit.  Krebsforsch.,  1906  (4),  417;  Muller,  Cent.  inn. 
Med.,  1909  (30),  89. 

90  Riforma  med.,  1903  (19),  1037. 

"  Deut.  Arch.  klin.  Med.,  1902  (72),  415. 


CHAPTER  IV 

THE    CHEMISTRY  OF  BACTERIA  AND   THEIR 
PRODUCTS 

STRUCTURE  AND  PHYSICAL  PROPERTIES^ 

In  structure,  as  in  nearl}'  all  other  respects,  bacterial  cells  stand 
intermediate  between  the  cells  of  ordinary  plant  and  animal  tissues. 
Their  cell  wall  seems  to  be  generally  more  highly  developed  than  that 
of  animal  cells,  and  less  so  than  the  wall  of  most  plant  cells.  The  much 
vexed  question  as  to  the  existence  or  non-existence  of  a  nucleus  seems 
to  be  best  answered  by  Zettnow,  who  considers  that  the  portion  of  the 
bacterial  cell  usually  made  evident  bj^  ordinary  staining  methods  con- 
sists of  a  mixture  of  nuclear  substance  (chromatin)  with  non-chromatic 
substance  {end o plasm) ;  the  outer  membrane,  which  requires  special 
methods  for  its  satisfactory  demonstration,  consists  of  a  modified 
cytoplasm  {ectoplasm).  Some  bacteria  consist  chiefly  of  chromatin 
(e.  g.,  vibrios),  but  the  proportion  of  the  different  elements  varies 
greatly,  not  only  in  different  varieties,  but  also  in  the  same  variety 
under  different  conditions.  The  fact  that  the  chromatin  is  not  aggre- 
gated into  the  usual  nuclear  form  may  be  ascribed  to  the  low  stage  of 
development  reached  by  bacteria  in  the  scale  of  evolution;  or,  as 
Vejdovosky  has  suggested,  to  the  extremely  rapid  rate  of  cell  division 
in  the  bacteria  which  prevents  the  chromatin  from  appearing  in  the 
resting  stage  which  a  nucleus  constitutes.  Finer  structures  within 
the  bacterial  cell  have  as  yet  been  only  imperfectly  discerned. 

The  thickness  of  the  ectoplasm  varies  greatly  even  in  the  same 
species,  being  generally  greatest  in  older  cultures.  In  some  forms 
the  ectoplasm  may  constitute  one-half  of  the  total  mass  of  the  cells. 
The  capsule  seems  to  arise  through  a  swelling  of  the  ectoplasm,  and 
is  probably  present  in  at  least  a  rudimentary  stage  in  all  bacteria 
(Migula). 

Plasmolysis  and  Plasmoptysis. — Under  conditions  of  altered 
osmotic  pressure  the  bacterial  cell  behaves  quite  similarlj^  to  the  plant 
cell. 2     If  placed  suddenly  in  a  solution  of  higher  osmotic  pressure  than 

1  In  this  chapter  references  will  not  generally  be  given  that  can  be  found  by 
consulting  Kolle  and  Wassermann's  Handbuch.  A  general  consideration  of  the 
Biology  of  the  Bacteria,  including  references  to  the  effects  of  light,  heat,  osmotic 
pressure,  etc.,  is  given  by  Miiller,  Ergb.  der  Physiol.,  190-1  (4),  138;  concerning 
their  chemistry  see  H.  Fischer,  Lafar's  Handbuch  der  Technischen  Mykologie, 
1908  (1),  222. 

''Literature,  see  Gotschlich,  Kolle  and  Wassermann's  Handbuch,  vol.  1. 

101 


102  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

the  one  in  which  it  has  been,  the  cell  contents  shrink  away  from  the  cell 
wall  {plasmolysis)  indicating  that  there  exists  a  semipermeable  mem- 
brane through  which  water  passes  more  rapidly  than  salts.  If  the 
change  in  osmotic  pressure  is  gradual,  the  bacteria  accomodate  them- 
selves to  it  by  the  slow  diffusion  of  the  salts  through  the  cell  membrane, 
indicating  that  it  is  not  absolutely  semipermeable.  Different  bac- 
teria behave  differently,  some  bacteria  not  being  plasmolyzed  by 
solutions  that  plasmolyze  others.  As  a  rule,  old  bacteria  plas- 
molyze  more  rapidly  than  young,  and  in  some  varieties  there  seems  to 
be  a  spontaneous  plasmolysis,  to  which  has  been  attributed  the  irregular 
staining  of  diphtheria  and  tubercle  bacilli,  the  polar  staining  of  plague 
bacilli,  etc.  Plasmolysis  occurs  only  in  living  bacilli,  but  does  not  nec- 
essarily cause  death.  The  Gram-staining  bacteria  cannot  generally 
be  plasmolyzed,  and  contain  more  water. ^ 

When  bacteria  pass  from  solutions  of  higher  osmotic  concentration 
into  solutions  of  lower  concentration,  the  phenomenon  of  plasmoptysis 
is  produced.  The  cell  contents  swell  until  the  cell  wall  gives  way  at 
some  point,  and  then  exude  as  glistening  drops,  which  may  become 
detached  from  the  wall  and  escape  free  into  the  fluid.  Plasmoptysis 
is  shown  best  by  bacteria  that  have  been  grown  on  salt-rich  media 
before  being  placed  in  the  salt-free  fluid.  Not  all  varieties  of  bacteria 
can  be  made  to  undergo  this  change,  depending  probably  upon  the 
degree  of  permeability  of  their  cell  membranes  for  salts.  The  ex- 
posure of  the  naked  cell  contents  to  the  hypotonic  fluid  outside  the 
cells  makes  plasmoptysis  more  serious  for  bacterial  life  than  plasmo- 
lysis, but  how  often,  if  ever,  either  process  plays  a  part  in  the  resistance 
of  infected  animals  against  bacteria  is  unknown.  The  resistance  of 
bacteria  to  direct  pressure  is  striking;  spore  bearers  may  not  be  killed 
under  direct  pressure  of  12,000  atmospheres  for  14  hours,  and  non- 
spored  bacteria  resist  3,000  but  not  6,000  atmospheres.^ 

Chemotaxis.^ — Just  as  with  unicellular  animal  organisms,  bacteria 
respond  to  chemotactic  influences,  in  general  being  attracted  bj''  sub- 
stances favorable  for  food,  such  as  peptone,  amino  acids, ^  dilute  potas- 
sium salts,  etc.,  and  being  repelled  by  harmful  substances,  such  as 
strong  acids  and  alkalies.  Attempts  have  been  made  to  separate 
different  organisms  in  mixed  cultures  by  means  of  their  response  to 
chemotaxis,  but  without  striking  success.  It  is  possible  that  chemo- 
taxis  may  play  a  part  in  the  localization  of  bacteria  from  the  blood 
stream  in  favorable  localities,  just  as  leucocytes  arc  attractd  to  points 
of  injury,  but  this  has  not  been  demonstrated.  (The  chemotactic 
influence  of  bacteria  upon  leucocytes  is  discussed  in  Chapter  xi.) 

3  Nioolle  and  Alilairc,  Ann.  Inst.  Pasteur,  1009  (23),  547. 
*Larfc'on,  Hartzell  and  Diehl,  Jour.  Infect.  I)is.,  lOlS  (22),  271. 
^  Concerning  the  adsorption  of  l)acteria  sec  Bechhold,  Kolloid-Zcitsrhr.,  1918 
(23),  35. 

"  Pringsheiin  and  Ernst,  Zcit.  pliysiol.  Clieni.,  19U)  (97),  17(). 


COMPOSITION  OF  BACTERIA  103 


CHEMICAL  COMPOSITION 


This  varies  greatly,  not  only  between  different  species,  but  even 
in  the  same  species  grown  on  different  media ;^  in  this  respect  bacteria 
are  much  more  modified  by  their  environment  than  are  higher  or- 
ganisms. On  the  other  hand,  they  can  develop  in  solutions  containing 
only  a  few  of  the  simplest  organic  and  inorganic  compounds  and  syn- 
thesize the  complex  components  of  their  cells,  as  well  as  enzymes, 
toxins,  pigments.^  They  usually  contain  between  80  and  90  per  cent, 
of  water.  Grown  on  a  salt-rich  medium  they  yield  much  ash;  grown 
on  a  peptone-rich  medium  they  contain  much  protein;  grown  on  a  fat- 
rich  medium  they  contain  much  material  solulile  in  ether.  Cholera 
vibrios  grown  on  a  bouillon  medium  contained  69.25  per  cent,  of  pro- 
tein, and  25.87  per  cent,  of  ash,  whereas  the  same  organism  grown  on 
Uschinsky's  medium,  which  contains  no  proteins  but  only  various 
simple  chemical  compounds,  contained  but  35.75  per  cent,  of  protein 
and  13.7  per  cent,  of  ash  (Cramer).  Even  in  the  same  medium  two 
different  strains  of  the  same  organism  may  show  equally  great  dif- 
ferences: Two  strains  of  cholera  vibrios  grown  on  the  same  medium 
showed  respectively  65.63  per  cent,  and  34.37  per  cent,  of  protein. 
It  is  evident,  therefore,  that  quantitative  analyses  of  bacteria  show 
nothing  as  to-  their  nature,  and  on  account  of  the  extreme  limits  of 
their  variation  are  practically  valueless.  The  specific  gravity  of  bac- 
teria, generally  between  1.12  and  1.345,  also  varies  wdth  media  and 
age.^     In  an  electric  field  they  move  towards  the  anode. ^"^ 

Qualitatively  the  variations  are  not  so  great — all  bacteria  contain 
proteins,  lipoid  substances,  and  salts,  of  which  phosphates  are  most 
prominent  in  the  ash.  The  character  of  the  proteins  and  fats  of 
bacteria  grown  on  peptone  bouillon  is  quite  the  same  as  when  they 
are  grown  on  protein-free  media.''  The  older  analyses  of  bacterial 
constituents  are  of  little  value.  Recent  studies  prove  that  the  chief 
constituent  of  the  cell  contents  is  a  true  nucleoprotein  (Iwanoff^^)  con- 
taining some  sulphur  and  iron;  probably  many  of  the  "pyogenetic  pro- 
teins," "bacterial  toxalbumins,"  "bacterial  caseins"  of  earlier  investi- 
gators are  true  nucleoproteins.^^  The  stainable  substance  of  anthrax 
bacilli  behaves  as  if  it  were  a  chromatin,  while  the  spores  resemble 

^  See  Dawson,  Jour.  Bact.,  1919  (4),  133. 

^  Concerning  fundamentals  of  nutrition  of  bacilli  see  Koser  and  Rettger,  Jour. 
Infect.  Dis.,  1919  (24),  301;  Long,  Amer.  Rev.  Tuberc,  1919  (3),  86. 
9  Stigell,  Cent.  f.  Bakt.,  1907  (43),  487. 

'"  Buxton,  Zeit.  physikal.  Chem.,  1906  (57),  47;  Girard  and  Audubert,  Compt. 
Rend.  Acad.  Sci.,  1918  (167),  301.  Concerning  the  decirical  condxiclivity  of 
bacteria  see  Thornton,  Proc.  Royal  Soc,  London,  Sec.  B.,  1913  (85),  331. 

"  Tamura,  Zeit.  physiol.  Chem.,  1913  (88),  190. 

'-  Hofmeister's  Beitr.,  1902  (1),  524;  bibliography  by  Lustig,  KoUe  and  Wasser- 
mann'8  Handbuch,  1913  (ii),  1362. 

' '  The  purity  of  many  of  the  preparations  worked  with  as  bacterial  nucleopro- 
teins,  is  very  doubtful.     (See  Wells,  Zeit.  Immunitat.,  1913  (19),  599.) 


104  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

linin  (Rozicka).^^  The  predominance  of  nuelein  compounds  is  shown 
by  Ruppel's  summary  of  the  composition  of  dried  tubercle  baciUi, 
namely, in  per  cent.,tuberculonucleic  acid,  8.5;  nucleo-protamine,  24.5; 
nucleo-protein,  26.5;  fatty  matter,  26.5;  inorganic,  9.2;  insoluble 
"proteinoid"  residue,  8.3.  In  a  water  bacillus  Nishimura  found 
xanthine,  guanine,  and  adenine,  indicating  the  presence  of  nucleo- 
protein;  others  have  found  that  bacterial  nucleoproteins  split  off 
pentoses,  as  do  the  nucleoproteins  of  higher  cells.  If  it  is  true  that 
bacterial  nucleo-proteins  contain  pentose  it  ranks  them  with  the  plant 
nucleo-proteins,  for  animal  nucleic  acids  contain  hexose.  On  the  other 
hand,  Levene  found  in  bacterial  nucleic  acid  the  pyrimidines,  thj-mine 
and  uracil,  which  are  respectively  characteristic  of  animal  and  vege- 
table nucleic  acids.  Mary  Leach^^  found  evidence  that  the  colon 
bacillus  is  largely  made  up  of  nuelein  or  glyco-nucleoproteins,  but 
contains  no  cellulose.  Other  proteins,  namely,  globulins  and  nucleo- 
albumins,  have  also  been  described  as  constituents  of  the  bacterial 
plasma. 

The^complete  amino-acid  content  of  bacterial  protein  does  not  seem 
to  have  been  worked  out,  although  the  workers  in  Vaughan's  labora- 
tory have  identified  many  of  the  usual  amino-acids  of  proteins  among 
the  products  of  hydrolysis  of  bacteria. ^^  Analysis  of  B.  mesentericus 
shows  it  to  be  deficient  in  diamino-acids,  tyrosine,  glycine,  and  to 
contain  16.6  per  cent,  of  glutamic  acid."  Tamura^^  found  phenyl- 
alanine and  valine  high  in  tubercle  bacilli  and  very  low  in  B.  diph- 
therice,  in  which  tyrosine  is  more  abundant.  In  an  azobacterium, 
lysine  has  been  found  especially  abundant.'^  Cystine  has  been 
lacking  in  several  analyses.  Tamura^°  also  found  that  bacteria  can 
synthesize  from  simple  nonprotein  media  the  purines,  phosphatids  and 
the  typical  proteins  containing  the  aromatic  amino-acids.  This  syn- 
thetic activity  of  bacteria,  in  view  of  the  large  quantity  of  bacterial 
substances  in  feces,  may  possibly  be  of  importance  in  metabolism 
studies,  leading  to  erroneous  conclusions  as  to  utilization  or  sjmthesis 
of  proteins  by  the  subject.^'  In  common  with  other  forms  of  cellular 
life,  bacteria  require  certain  specific  substances,  "vitamins,"  to  permit 
of  their  growth  ;22  also  they  produce  substances  with  the  value  of 
vitamins.-^ 

1^  Arch.  Entwicklungsmk.,  1906  (21),  306. 

/^  Jour.  Biol.  Chem.,  1906  (1),  463.  Full  bibliography  on  Chemistry  of  Bac- 
teria. See  also  Vaughan,  "Protein  Split  Products  in  Relation  to  Immunity  and 
Disease,"  Philadelphia,  1913. 

'« See  Wheeler  Jour.  Biol.  Chem.,  1909  (6),  509. 

'^  Horowitz- Wiassowa,  Arch.  Sci.  Biologique,  1910  (15),  40. 

'8  Zeit.  phvsiol.  Chem.,  1913  (87),  85;  1914  (89),  289. 

19  Omelian.sky  and  Sieber,  Zeit.  physiol.  Chem.,  1913  (88),  445. 

20  Zeit.  physiol.  Chem.,  1913  (88),  190. 

2'  Osborne  and  Mendel,  Jour.  Biol.  Chem.,  1913  (18),  177. 
"See  Davis,  Jour.  Infect.  Dis.,  1917  (21),  392;  Kligler,  Jour.  Exp.  Med.,  1919 
(30),  31. 

"  Pacini  and  Russell,  Jour.  Biol.  Chem.,  1918  (34),  43. 


BACTERIAL  CARBOHYDRATES  Ai\D  LII'IXS  105 

The  slimy  material  produced  in  cultures  by  some  varieties  of  bac- 
teria is,  at  least  for  certain  forms,  a  body  closely  related  to  or  identi- 
cal with  true  mucin, ^^  but  in  certain  cases  {B.  radicicola)  it  is  a  gum 
related  to  the  dextrans  and  free  from  nitrogen  (Buchanan). ^^  Tu- 
bercle bacilli  grown  for  many  years  on  artificial  media  may  produce 
a  true  mucin  (Weleminsky).-^  Heim"  considers  that  anthrax  bacilli 
also  produce  mucin.  Some  nonpathogenic  bacteria  contain  granules 
of  sulfur  in  their  protoplasm,  and  others  have  noteworthy  quantities 
of  iron  in  the  sheath. 

Bacterial  Carbohydrates. — -The  earlier  descriptions  of  cellulose  or 
hemicellulose  in  the  cell  membrane  of  bacteria  have  been  contested. ^^ 
Numerous  investigators  have  reported  that  the  insoluble  bacterial  cell 
wall  consists  chiefly  of  chitin,  which  on  being  split  with  acids  yields 
80  to  90  per  cent,  of  the  nitrogenous  carbohydrate,  glucosamin.^'^  The 
distinction  is  a  verj^  important  one,  since  cellulose  is  a  typically  vege- 
table product,  while  chitin  is  equally  typically  animal  in  origin,  being 
found  chiefly  in  the  shells  of  lobsters  and  crabs,  the  wings  and  cover- 
ings of  flies,  beetles,  etc.  Chitin  seems  to  be  a  polymeric  form  of 
glucose-amine,^"  an  amino-carbohydrate,  just  as  cellulose  is  a  polymer 
of  a  simpler  carbohydrate.  Other  carbohydrates  seem  to  be  scanty  in 
the  bacterial  cell,  but  Tamura^^  does  not  accept  the  chitinous  nature 
of  bacterial  carbohydrate,  finding  in  tubercle  and  diphtheria  bacilli  a 
hemicellulose,  apparently  a  pentosan  yielding  1-arabinose  on  hydro- 
lysis. Wester^^  found  no  chitin  in  several  varieties  of  bacteria,  and 
cellulose  only  in  B.  xylinum;  he  therefore  considers  it  probable  that 
bacterial  cell  walls  do  not  alw^ays  consist  of  the  same  substance.  Cra- 
mer could  find  no  glucose  in  any  variety,  although  there  are  some  bac- 
teria that  contain  material  reacting  like  starch  with  iodin.  Levene,^^ 
however,  found  in  B.  tuberculosis  a  substance  with  some  of  the 
properties  of  gl3^cogen. 

Bacterial  Fats. — By  staining  methods,  fats  have  been  recognized  in 
many  species,  and  by  extraction  with  fat  solvents  lecithin,  cholesterol, 
simple  fats,  and  specific  bacterial  fats  have  been  isolated;  this  is  par- 
ticulary  true  of  B.  tuberculosis.^^     Numerous  studies  of  these  fats  of 

-*  Rettger,  Jour.  Med.  Research,  1903  (10),  101. 

"  Cent.  f.  Bakt.,  II  Abt.,  1909  (22),  371. 

-^  Berl.  klin.  Woch.,  1912  (49),  1320. 

"  Miinch.  med.  Woch.,  1904  (51),  426. 

28  However,  Dreyer  (Zeit.  ges.  Brauw.,  1913  (36),  201)  states  that  the  cell 
wall  of  yeasts  contains  a  hemicellulose  and  a  manno-dextran.  See  also  Kozniewski, 
Zeit.  physiol.  Chem.,  1914  (90),  208. 

=9  See  Viehofer.  Ber.  Deut.  Chem.  Ges.,  1912  (30),  443. 

'"  Morgulis  states  that  chitin  consists  of  two  parts,  one  containing'all  the 
glucose  and  amino  groups,  the  other  being  a  stable  nitrogenous  compound  yielding 
no  glucose.     (Science,  1916  (44),  S66.) 

"  Zeit.  phvsiol.  Chem.,  1914  (89),  304. 

22  Pharm.  Weekblad,  1916  (53),  1183. 

"Jour.  Med.  Research,  1901  (6),  135. 

^*  See  Camus  and  Pagniez,  Compt.  Rend.  Soc.  Biol.,  1905  (59),  701. 


106  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

B.  tuberculosis  have  been  made^^  and  by  using  different  extractives, 
from  20  to  40  per  cent,  of  the  entire  weight  of  the  bacilh  has  been 
found  sokible  in  fat  solvents.  Kreshng  found  that  the  substance 
soluble  in  chloroform  had  the  following  composition: 

Free  fatty  acid 14.38  per  cent. 

Neutral  fats  and  fatty  acid  esters 77.25  per  cent. 

Alcohols  obtained  from  fatty  acid  esters 39.10  per  cent. 

Lecithin 0. 16  per  cent. 

Substances  soluble  in  water 0.73  per  cent. 

Bulloch  and  Macleod  found  that  ethereal  extracts  did  not  contain 
the  acid-fast  substance  which  they  consider  to  be  a  wax-like  alcohol, 
soluble  in  hot,  but  insoluble  in  cold  absolute  alcohol  or  in  ether. 
The  simple  fats  seem  to  be  formed  by  oleic,  isocetinic,  and  myristinic 
acids,  and  there  is  some  lauric  acid  in  the  form  of  a  soap.  Kozniewski'® 
obtained  what  seemed  to  be  a  lauric  acid  ester  of  a  dodecyl-alcohol, 
and  Biirger^^  attributes  the  odor  of  tubercle  bacilli  to  the  presence  of 
salicylic  aldchj^de.  Cholesterol  could  not  be  found  in  tubercle, 
diphtheria  and  other  bacteria  examined  by  Tamura,  although  there 
probably  are  lipochromes  giving  the  cultures  their  color. ^^  There  is 
still  much  disagreement  as  to  whether  the  acid  fastness  of  tubercle 
bacilli  depends  upon  waxes,  alcohols,  fatty  acids,  or  lipoid-protein 
compounds. ^^  It  must  be  admitted  that  a  high  content  of  fatty  ma- 
terials is  regularly  present  in  acid-fast  bacilli;  thus,  in  an  acid-fast 
bacillus  isolated  from  leprous  lesions,  34.7  per  cent,  of  fats,  fatty  acids 
and  cholesterol,  and  1.7  per  cent,  of  lecithin  were  found  by  Gurd  and 
Denis. ^^  Miller"*!  attributes  the  unstained,  spore-like  areas  of  tubercle 
bacilli  to  oleins,  as  bacilli  grown  on  olive  and  sperm  oil  show  a  marked 
decrease  in  acid  fast  areas. 

Tamura*^  states  that  the  phosphatids  of  B.  tuberculosis  and  a 
saphrophyte  exaniinetl  by  him  were  not  lecithin  but  a  diaminophos- 
phatid,  although  diphtheria  bacilli  seemed  to  contain  lecithin.'*^  He 
found  in  both  a  high  molecular  alcohol,  "mykol,"  to  which  he  ascribes 
acid-  and  Gram-fastness.  In  a  Gram-negative  bacillus**  he  found 
lecithin,  but  no  cholesterol  or  mykol.  Apparently  the  fats  of  tubercle 
bacilli  resemble  in  character  and  complexity  the  "waxes"  of  j)lants 
(Burger), ^^  which  are  called  "ccrolii)oids"  b}^  Czajiok.  By  growing 
tubercle  bacilli  on  suitable  media  they  can  l)o  made  1o  lose  their  acid- 

"^  For  literature  see  BuUocli  and  Maclood,  .Tour,  of  lly^fiene,  1004  (4),  1. 

^*  An/eifzicr  d.  Akad.  Wiss.  KraUau,  Matli.-naturwiss  Kl.,  1912,  p.  942. 

"  liiocliein.  Zeit.,  191(>  (7S),  155. 

"  I'anzer  (Zcil.  jjliysiol.  CIhmii.,  1912  (78),  414)  could  not  demonstrate  cho- 
lesterol in  tiihorcle  Invcilli  l)ut  did  lind  a  small  amount  of  some  substance  imitinp; 
with  (lij:;it()nin. 

"•Sec  Camus  and  Pnnniez,  Pre.sse  Med.,  1907  (15),  05;  Deyke,  Munch,  mcd. 
Woch.,  1910  (57),  (VM. 

"Jour.  J';\i)er.  Med.,  1911  (11),  COG. 

■"  .Jour,  i'utii.  and  linel.,  191(1  (21),  11. 

^^Zcit.  i)iivsi(>l.  Clieiii.,  1913  (S7),  S5. 

"//;i(/.,  1911  (S9),  2,S9. 

**  Ilnd.,  1914  (90),  2S0. 


BACTERIAL  LII'INS  107 

fast  property,  although  still  Gram-positive  (Wherry).''^  The  observa- 
tion of  Miss  Sherman, ^^  that  tubercle  bacilli  are  almost  absolutely 
impermeable  to  fat-soluble  dyes  which  stain  their  isolated  fats  well, 
and  her  corroboration  of  Benians'  demonstration  that  acid-fastness 
depends  on  the  integrity  of  the  bacillary  envelope,  make  the  role  of 
the  fatty  substances  uncertain.  The  high  content  in  unsaturated  fatty 
acids  gives  acid-fast  bacteria  a  high  antitryptic  power,  which  may  be 
concerned  in  the  defense  of  the  bacteria  and  also  in  the  persistence  of 
caseous  material  in  tubercles  (Jobling  and  Petersen).*^  The  oily 
material  obtained  by  extracting  tubercle  bacilli  with  cold  ether  is 
non-toxic,  while  the  waxy  material  extracted  with  hot  alcohol  produces 
foreign  body  tubercles  (Morse  and  Stott).*^ 

By  staining  with  Sudan  III,  Sata^^  demonstrated  fats,  not  only  in 
the  acid-fast  bacilli,  but  also  in  anthrax,  Staphylococcus  aureus,  B. 
mucosus,  and  actinomyces;  but  not  in  diphtheria,  pseudo-diphtheria, 
plague,  cholera,  and  chicken  cholera  bacilli,  or  in  members  of  the 
colon  group. ^^  Only  a  few  bacteria  form  fat  on  agar  free  from  gly- 
cerol, but  potato  is  a  favorable  medium.  Ritchie^^  obtained  positive 
fat  staining  in  B.  diphtherice  and  anthracis,  but  not  in  S.  pyogenes 
aureus  or  M.  tetragenus,  although  these  last  forms  contain  chemically 
demonstrable  lipins.  Analyses  of  different  bacteria  show  a  relatively 
low  content  of  lipins  as  compared  with  tubercle  bacilli,  varying  from 
1.7  per  cent,  in  B.  subtilis  to  8.5  per  cent,  in  staphylococci  (Jobling  and 
Petersen). ^2  However,  the  degree  of  unsaturation  of  the  fatty  acids 
is  less  with  tubercle  bacilli  than  with  other  bacteria  examined  by 
these  authors.  Extensive  studies  of  bacterial  fat  stains  are  reported 
by  Eisenberg,^^''  but  practically  nothing  is  known  of  the  character  of 
the  fatty  or  lipoid  constituents  of  bacteria  outside  the  acid-fast  group. 

Spores  differ  from  their  parent  bacteria  in  containing  a  much  greater  propor- 
tion of  the  soUd  constituents  and  less  water.  In  molds  Drymont  found  that  the 
spores  contained  over  60  per  cent,  of  dry  substance,  and  almost  all  the  water  was 
so  held  as  to  resist  drj'ing  by  temperatures  below  boiling;  the  drj'  substance  is 
very  rich  in  protein  and  poor  in  salts.  As  the  spores  may  lose  their  chromatin 
content  without  loss  of  capacity  to  propagate,  it  would  seem  that  this  is  not  a 
nuclear  chromatin  but  merely  a  reserve  food  supply.^'  The  wall  of  the  spore  con- 
sists of  a  ''cellulose-like  "  substance  and  a  very  hygroscopic  extractive  matter.  The 
great  resistance  of  spores  to  drying  and  to  heat  can  be  readily  understood  in  view 
of  these  facts.  They  contain,  and  perhaps  secrete,  active  enzymes  (Effront).*' 
Flagella  also  seem  to  be  composed  of  a  relativelj'  condensed  protein. 

"Jour.  Infect.  Dis.,  191.3  (13),  144. 

"  Jour.  Infect.  Dis.,  1913  (12),  249. 

^' Jour.  Exp.  Med.,  1914  (19),  239. 

«  Jour.  Lab.  Chn.  Med.,  1916  (2),  159. 

«  Cent.  f.  allg.  Path.,  1900  (11),  97. 

^^  Auclair  (Arch.  M;'d.  Exper.,  1903  (15),  725)  contends  that  the  ether  and 
chloroform  extracts  of  many  pathogenic  bacteria  contain  important  toxic  sub- 
stances. Holmes  (Guy's  Hosp.  Reports,  1905  (59),  155)  states  that  injection 
of  fatty  acids  from  tubercle  bacilli  into  rabbits  causes  a  lymphocytosis. 

6'  Jour.  Pathol,  and  Bact.,  1905  (10),  334. 

"Jour.  Exp.  Med.,  1914  (.20),  456. 

52"  Virchow's  Archiv.,  1910  (199),  502. 

"  Ruzicka,  Cent.  f.  Bakt.,  1914  (41),  641. 

"  Mon.  sc.  Quesneville,  1907,  p.  81. 


108  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

Staining  Reactions. — The  staining  reactions  of  bacterial  cells 
are  much  as  if  the  bacteria  consisted  entirely  of  chromatin,  so  that  at 
one  time  the  theory  prevailed  that  bacteria  consisted  merely  of  a 
nucleus  and  a  cell  wall,  without  any  true  cytoplasm.  The  demon- 
stration of  abundant  nuclcoprotein  in  the  contents  of  bacterial  cells 
explains  their  staining  affinity  for  basic  anilin  dyes.  Owing  to  some 
unknown  differences  in  composition,  not  all  bacteria  are  stained 
equally  well  by  the  same  basic  dyes."  Although  the  staining  of  bac- 
teria depends  upon  a  chemical  reaction  between  the  nucleoproteins  and 
the  basic  dye,  yet  the  combination  is  not  usually  a  firm  one,  being 
readily  broken  by  weak  acids  in  most  cases.  That  the  decolorization 
of  bacteria  depends  upon  dissociation  of  the  dye-protein  compound 
is  shown  by  the  fact  that  absolutely  water-free  alcohol  will  not  de- 
colorize dry  bacteria,  nor  do  water-free  alcoholic  solutions  of  dyes 
stain  dehydrated  bacteria.  There  seems  to  be  a  marked  difference  in 
the  accessibility  of  dead  and  living  bacteria  to  stains;  thus,  only  dead 
bacteria  stain  with  AgNOs." 

Gram's  Method^'  of  staining  has  been  ascribed  to  the  formation  of  an  iodin- 
pararosanilin-protein  compound  which  is  not  easily  dissociated  by  water  in  the 
case  of  bacteria  that  stain  by  this  method,  and  which  is  readily  dissociated  and 
dissolved  out  in  the  case  of  bacteria  that  do  not  retain  the  stain.  Only  para- 
rosanilin  dyes  (gentian  violet,  methyl  violet,  victoria  blue)  form  such  combinations, 
the  rosanilin  dyes  not  being  suitable.^'*  It  is  probable,  especially  from  the  obser- 
vations of  Deussen,  that  the  nucleoproteins  are  the  essential  cell  constituents,  and 
other  cells  than  bacteria  {'.<;..  sperm)  may  be  Gram-positive. 

The  relation  of  bacterial  protein  to  Gram  staining  is  shown  by  the  fact  that 
trypsin  will  digest  killed  bacteria  which  are  Gram-negative,  but  not  Gram-positive 
forms;  gastric  juice  attacks  only  a  few  Gram-positive  bacteria. ^'^  They  are  also 
more  resistant  to  alkalies,  1  per  cent.  KOH  dissolving  only  the  Gram-negative 
bacteria.  Brundy'"  considers  that  they  are  more  permeable  to  iodin,  so  that  a 
more  central  iodin-dye  precipitate  occurs,  and  Eisenberg*''  suggests  that  lipoid- 
protein  compounds  in  the  surface  are  important,  in  support  of  which  is  the  obser- 
vation that  ether  extraction  of  staphylococci  renders  them  negative  to  Gram's 
method,  while  colon  bacilli  treated  with  lecithin  become  positive.*^'-  Jobling  and 
Petersen^^  have  also  found  the  lipoids  of  Gram-positive  bacteria  more  resistant 
to  extraction  by  fat  solvents  than  lipoids  of  Gram-negative  bacteria,  and  Tamura^' 
found  that  the  lipoid  extract  contains  the  bacterial  element  responsiVile  for  Gram 
staining.  The  first-named  authors  suggest  a  relation  between  the  high  content 
in  unsaturated  fatty  acids,  with  the  higli  affinity  for  iodin,  and  the  positive  Gram 
staining.  On  the  other  hand,  Ilottinger'''  attributes  (!ram  staining  solely  to  the 
degree  of  disjjersion  of  the  nucleo-proteins,  which  he  believes  \o  be  higher  in  the 

'*  The  presence  of  serum  interferes  with  staining,  probably  from  protective 
colloid  action  (Fleisher,  .lour.  Med.  Res.,  1017  (3()),  31.) 

'«  Nyfeldt,  Nordiskt  Med.  Arkiv,  1917  (^O),  1S4. 

"  Full  review  by  Deussen,  Zeit.  llyg.,  191S  (Sf)),  23r). 

'*  Any  metallic  iodid  may  be  substituted  for  Kl  (Leidv,  .lour.  Lab.  Clin.  Med., 
1919  (4),  3.04). 

'"  Hingers,  Schermann  and  Schrciber,  Zeit.  f.  Ilyg.,  1911  (10),  119;  ^^'einkoff, 
Zeit.  Imuiunilat.,  1912  (11),  1. 

""Cent.  f.  iiakt.,  ii  Abt.,  190S  (21),  62. 

«'  Cent.  f.  Hakt.,  1910  (.')(>),  193. 

«Mour.  rath,  and  Hact.,  1911  (Hi),  140. 

''Zeit.  i)livsiol.  Ghem.,  1914  (S9),  2S9. 

«'  Cent.  f.  iiakt.,  1910  (7(1),  3t)7. 


BACTERIAL  ENZYMES  109 

Gram-nepativc  forms.  Benians*^  has  found  that  crushed  Gram-positive  bacteria 
are  proni])tly  decolorized,  indicating  that  the  dye  and  the  cell  contents  do  not 
form  an  insoluble  comjiound,  but  that  the  l)ac"terial  cell  \vall  is  the  chief  factor  in 
detennininK  CIram  jiositiveness;  presumably  the  iodin  renders  the  cell  membrane 
impermeable  to  alcohol.  This  important  contribution  has  been  confirmed,  as 
far  as  the  staining  of  tubercle  bacilli  is  concerned,  by  Hope  Sherman,*"  who  corrob- 
orates the  finding  of  Benians  that  if  the  bacilli  are  not  intact  they  are  neither  acid 
fast  nor  (Irani  ])ositive.  The  same  is  true  of  yeast  cells  (Henrici)/'  but  Dcus^en 
states  that  press  juice  from  yeast  (Buchncr's  zymase)  contains  Gram-positive 
£;ranules. 

Bacterial  Enzymes^^ 

The  metabolic  processes  of  bacteria  seem  to  be  closely  dependent 
upon  enzyme  action,  just  as  with  higher  cells.  Liquefaction  of 
gelatin  is  a  familiar  example  of  the  enzyme  action  of  bacteria;  and 
since  the  filtered  cultures  of  liquefactive  bacteria  are  also  capable 
of  digesting  gelatin,  the  enzymes  are  evidently  excreted  from  the 
cells.  Dead  bacteria,  killed  by  thymol  or  by  other  antiseptics  that 
do  not  destroy  proteolytic  enzymes,  will  also  digest  gelatin.  Numer- 
ous investigations  have  established  the  wide-spread  occurence  of 
many  soluble  enzymes  both  in  bacteria  and  in  their  secretions,  indi- 
cating that  bacterial  cells  are  as  dependent  on  enzymes  for  the  pro- 
duction of  their  metabolic  activities  as  are  higher  types  of  cells,  and 
that  these  enzj^mes  are  not  only  present  as  intracellular  constituents, 
but  that  they  also  escape  from  the  cells.  Even  the  spores  contain 
active  enzymes. ^^  A  striking  property  of  bacteria  is  their  reducing 
power,  which  has  led  to  the  introduction  of  selenium  and  tellurium 
salts,  which  are  reduced  to  the  metals,  as  an  index  of  bacterial  life 
and  activity  (Gosio). 

The  diffusion  method  of  Wijsman,  or,  as  it  is  more  frequently 
called,  auxanogmphic  method  of  Beijerinck,  offers  a  relatively  simple 
means  of  detecting  the  presence  of  extracellular  bacterial  enzj-mes. 
Eijkman^"  in  particular  has  used  this  method,  which  consists  of  mix- 
ing agar  with  milk,  or  starch,  or  whatever  material  is  to  serve  as  the 
indicator  of  the  enzyme  action;  the  agar  is  then  inoculated  with  bac- 
teria and  plated  (or  else  the  bacteria  are  inoculated  as  a  streak  on  the 
surface  of  the  agar) .  About  each  colony  there  will  appear  a  zone  of 
clearing  in  the  medium  if  it  produces  enzymes  digesting  the  admixed 
substance.  By  this  means  Eijkman  found  that  all  bacteria  that 
produce  enzymes  digesting  gelatin  also  digest  casein,  and  those  that 
do  not  digest  gelatin  are  equally  without  effect  on  casein;  therefore, 
it  is  probably  the  same  enzyme  that  digests  both.     As  the  hemolytic 

65  Jour.  Path,  and  Bact.,  1912  (17),  199. 

66  Jour.  Infec.  Dis.,  1913  (12),  249. 
6^  Jour.  Med.  Res.,  1914  (30),  409. 

68  See  Fuhrmann  ("  Vorlesungen  iiber  Bakterienenzyme,"  Jena,  1907)  for  com- 
plete bibliography  to  that  date. 

65  Effront,  Mon.  sc.  Quesneville,  1907,  p.  81. 
"  Cent.  f.  Bakt.,  1901  (29),  841. 


110  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

action  of  bacteria  is  not  constantly  related  to  their  gelatin-dissolving 
property,  the  hemolysis  probably  is  produced  by  other  means  than 
the  proteolytic  enzymes. ^^  A  few  pathogenic  bacteria  (anthrax, 
cholera  and  some  strains  of  hemolytic  streptococci^^)  digest  starch" 
and  B.  pyocyaneus,  StapJnjlococcus  pyogenes  aureus,  and  B.  prodigiosus 
all  produce  fat-splitting  enzymes  demonstrable  by  tliis  method."'* 
B.  pyocyaneus,  Eijkman  found,  digested  elastic  tissue  readily,"  as  also 
did  a  bacillus  resembling  B.  subtilis  obtained  from  the  tissue  of  a  gan- 
grenous lung. 

Rennin  is  produced  by  many  bacteria,  as  is  shown  by  their  coagu- 
lating milk,  independent  of  any  acid  reaction,"  and  protease  from 
pyocyaneus  causes  "plastein"  formation  in  albumose  solutions  (Zak),^^ 
Bacteria  which  give  negative  results  by  the  plate  method  may  con- 
tain active  lipase  demonstrable  in  killed  bacteria  by  direct  action 
upon  fats  and  esters,  these  lipases  behaving  exactly  like  the  lipase  of 
animal  tissues  (Wells  and  Corper);"  staphylococcus  and  pyocyaneus 
are  more  actively  lipolytic  than  B.  coli,  B.  dysenierice  and  B.  tuber- 
culosis. Urease  seems  to  be  widespread."  Tubercle  bacilli  contain 
enzymes  resembling  lipase,  trypsin,  erepsin,  nuclease  and  urease, 
but  not  amylase,-  elastase  or  invertase.^° 

Schmailowitsch^^  stated  that  the  amount  and  nature  of  enzymes 
produced  by  bacteria  is  modified  by  the  amount  and  nature  of  their 
food,  but  Jordan  found  that  gelatinase  is  produced  by  bacteria  grow- 
ing on  non-protein  media;  he  failed  entirely  to  support  the  statement 
of  Abbott  and  Gildersleeve^^  that  bacteria  grown  on  gelatin  produce 
much  more  active  gelatin-dissolving  enzyme  than  do  bacteria  grown 
on  bouillon.  Diehl^^  found  that  bacteria  grown  on  media  containing 
no  organic  nitrogen  produce  no  proteolytic  enzymes,  and  the  enzyme 
content  of  bacteria  is  much  modified  by  the  composition  of  the  media, 
depending  on  the  character  of  the  amino-acids  present  rather  than  the 
proteins  themselves.     Jacoby'*'*  has  made  extensive  studies  on  the 

^'  See  Jordan,  Biol.  Studies  by  the  pupils  of  W.  T.  Sedgwick,  1906,  p.   124. 

"  Tongs,  Jour.  Aincr.  Med.  A.ssoc.,  1919  (73),  1277. 

'•'  In  relation  to  carbohydrate  enzymes,  the  extensive  studies  of  Kendall  (Jour. 
Biol.  Chem.,  1912,  vol.  12)  should  be  consulted.  He  emphasizes  especially  that  as 
a  rule  bacteria  ferment  carboliydrates  in  preference  to  attacking  proteins  when 
both  foodstuffs  are  available. 

"  See  Buxton  (American  Med.,  1903  (0),  137)  concerning  enzymes  of  numerous 

Dfl.ctiGri&i 

">  Cent.  f.  Bakt..  1903  (35),  I. 

"  Contradicted  by  DcWacle,  Cent.  f.  Bakt.,  1905  (39),  353. 
"  llofmeister's  Beitr.,  1907  (10),  287. 

"Jour,  infect.  Dis.,  1912  (11),  388;  literature  on  bacterial  lipases.  See  also 
Kendall." 

'"  See  Jacoby,  Biochem.  Zeit.,  1917  (80),  357. 

80  Corper  and  Sweany,  Jour,  liact.,  1918  (3),  129. 

«'  Wratschel)naja  Cazetta,  1902,  p.  52. 

«2.Jour.  Med.  Re.seareli,  1903  (10),  42. 

«Mour.  Infect.  Dis.,  1919  (21),  347. 

«♦  Biochem.  Zeit.,  1917  (83),  74. 


BACTERIAL  ENZYMES  111 

requirements  for  the  production  of  urease  on  Uschinski's  medium, 
and  finds  that  while  bacteria  will  grow  if  the  sodium  asparaginate  is 
present,  no  urease  is  formed  unless  leucine  is  also  added.  There  does 
not  seem  to  be  any  important  relation  between  enzyme  production 
and  pathogenicity.*^ 

In  general,  bacterial  proteolytic  enzymes  resemble  trypsin  more 
closely  than  they  do  pepsin,  acting  best  in  an  alkaline  medium;  but 
the  enzymes  extracted  from  bacterial  cultures  are  very  feeble  as  com- 
pared with  pancreatic  trypsin.  It  is  probable  that  there  are  several 
distinct  proteolytic  enzymes  in  bacterial  cells,  gelatinase  being  a  dis- 
tinct protease  (Jordan). ^"^  Abbott  and  Gildersleeve  found  that  the 
gelatin-dissolving  enzyme  of  bacteria  resists  a  temperature  of  100°  C. 
for  as  long  as  fifteen  to  thirty  minutes,  but  Jordan  found  that  the 
reaction  of  the  medium  modifies  greatly  this  heat  resistance.  Schmailo- 
witsch*^  states  that  some  bacteria  produce  an  enzyme  acting  in  acid 
medium  upon  gelatin  but  not  upon  albumin,  and  this  enzyme  carries  the 
digestion  only  as  far  as  the  gelatin-peptone  stage,  whereas  the  enzymes 
acting  in  an  alkaline  medium  carry  the  splitting  through  to  leucine, 
tyrosine,  etc.  Kendall  and  Walker*^  state  that  the  proteolytic  enzymes 
of  B.  proteus  are  not  formed  when  the  bacteria  have  enough  carbo- 
hj^drate  supplied  so  that  they  need  not  depend  on  proteins  for  their 
energy  requirements;  deaminization  is  independent  of  proteolysis 
and  represents  intracellular  enzjTne  action.  Plenge^^  suggests  that 
there  is  a  special  enzyme  digesting  nucleoproteins  (nuclease).  Bac- 
teria are  able  to  split  nucleic  acids  and  to  convert  amino-purines  into 
oxypurines,  but  they  do  not  carry  the  oxidation  to  uric  acid;  putre- 
factive bacteria  can  slowdy  destroy  uric  acid  (Schittenhelm),^''  and 
B.  coll  destroys  purines. ^^ 

Cacace^-  investigated  the  cleavage  products  of  gelatin  and  coagu- 
lated blood  when  digested  by  B.  anthracis,  Staph,  pyogenes  aureus, 
and  Sarcina  aurantiaca,  and  found  that  proteoses  and  peptone  are 
produced,  which  disappear  in  the  later  stages  of  digestion.  Rettger^^ 
found  leucine,  tyrosine,  tryptophane,  as  well  as  phenols,  skatole,  indole, 
aromatic  oxy-acids,  and  mercaptan,  among  the  products  of  bac- 
terial decomposition  of  egg-albumen  and  meat;  proteoses  and  pep- 
tones appear  in  the  early  stages,  but  later  disappear,  as  also  eventually 
do  the  leucine,  tj-rosine,  etc.     Choline  has  also  been  found  in  the 

85  Rosenthal  and  Patai,  Cent.  f.  Bakt.,  1914  (73),  406;  (74),  369. 

8«  Corroborated  by  Bertiau,  Cent.  f.  Bakt.,  1914  (74),  374. 

"  Abst.  in  Biochem.  Centr.,  1903  (1),  230;  see  also  DeWaele,  Cent.  f.  Bakt., 
1905  (39),  353. 

8*  Jour.  Infect.  Dis.,  1915  (17),  442.  See  also  Berman  and  Rettger,  Jour. 
Bact.,  1918  (3),  367. 

89  Zeit.  f.  physiol.  Chem.,  1903  (39),  190. 

90  Zeit.  physiol.  Chem.,  1908  (57),  21. 

»'  Siven,  Zeit.  phvsiol.  Chem.,  1914  (91),  336. 

92  Cent.  f.  Bakt.,  1901  (30),  244. 

93  Amer.  Jour,  of  Physiol.,  1903  (8).  284 


112  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

products  of  autolysis.^*  MoUiard^^  reports  that  Isaria  densa  produces 
large  masses  of  crystals  of  glycine,  even  when  grown  on  proteins  that 
contain  little  or  no  glycine. 

The  digestive  power  of  the  filtrates  of  cultures  and  of  killed  bacteria 
is  far  less  than  that  of  the  living  bacteria  (Knapp).^^  Streptococci 
digest  proteins  of  exudates  feebly,  staphjdococci  more  rapidl}-,  and 
colon  bacilli  are  still  more  active.  He  could  find  no  relation  between 
the  proteolytic  power  of  the  bacteria  and  the  severity  of  the  infection 
from  which  they  came.  Staphylococci  can  cause  coagulation  of  plasma 
and  then  dissolve  the  coagulum,  showing  the  presence  of  two  enzymes, 
staphylokinase and  fibrinolysi7i  (KMnschmidt) .^"^  Sperry  and  Rettger,^^ 
however,  found  that  even  the  most  actively  putrefactive  bacteria  are 
unable  to  attack  or  grow  upon  carefullj^  purified  proteins,  although 
the  presence  of  small  amounts  of  amino  acids  or  other  available  nu- 
trient makes  the  proteins  available  to  the  bacteria;  apparently  they 
must  have  some  nutrient  more  available  than  intact  protein  molecules 
to  enable  them  to  grow  sufficiently  to  produce  enough  free  enzymes  to 
attack  the  proteins.  By  virtue  of  their  proteolytic  enzymes,  filtrates 
of  bacteria  that  liquefy  gelatin  also  can  digest  hardened  liver,  kidney 
and  other  tissue  elements  in  vitro  the  changes  resembling  those  of 
necrobiosis.^^ 

Oxidizing  Enzymes. — Catalase  is  demonstrable  in  bacteria,  the  anaerobic 
forms  showing  the  least  activity  (Rywosch),'  but  practically  no  species  is  entirely 
inactive  (.Joi'nsj ;'  it  may  exist  as  either  endo-  or  ecto-enzyme.  B.  pi-oteus  synthe- 
sizes catalase  even  when  grown  on  a  simple  sj^nthetic  medium  containing,  besides 
inorganic  salts,  sodium  lactate  and  alanine  or  aspartic  acid  (Jacob^-).^  Certain 
bacteria  and  actinomyces  exhibit  oxidative  effects,  resembling  tyrosi7iase,  but  such 
an  enzyme  could  not  be  extracted  by  Lehmann  and  Sano.''  Tsudji,*  however, 
not  only  observed  oxidation  of  tyrosine,  but  states  furthermore  that  proteus  pro- 
duces always  a  d-oxyacid  product  and  subtilis  a  1-oxyacid  type,  regardless  of 
whether  they  have  oxidized  d-,  1-,  or  dl-tyrosine. 

Immunity  against  bacterial  enzymes  may  be  secured  as  it  is  against 
other  enzymes.  Abbott  and  Gildcrsleeve^-  found  that  by  injections 
into  animals  of  proteolytic  bacterial  filtrates  which  were  only  slightly 
toxic,  the  serum  of  the  animals  acquired  a  slight  but  specific  increase 
in  resistance  to  the  proteolytic  enzymes  of  the  filtrates.^  Normal 
serum   contains    a   certain    amount   of    enzyme-resisting    substance. 

»^  Kutscher  and  Lohmann,  Zcit.  phvsiol.  Chcm.,  1903  (39),  313. 

»''Compt.  Rend.  Acad.  Sci.,  1918  (167),  7S(i 

"oZeit.  f.  Ileilk.  (Chir.  Abt.),  1902  (23),  230. 

0'  Zcit.  Immunitiit.,  1909  (3),  510. 

•■"•.four.  Biol.  Chem.,  1915  (20),  445;  Jour.  Hact.,  1910  (1),  15. 

»'■'  Bittrolff,  Zicgler's  Beitr.,  1915  (00),  337. 

•  Cent.  f.  Bakt.,  1907  (44),  295. 
2  Arcli.  f.  Ilvg.,  190.S  (07),  134. 

=•  Biocliem.  Zcit.,  191S  (SS),  35  and  (89),  350. 

<  Arcli.  f.  llyg.,  190S  (()7),  99. 

'  Acta  Schoiac  Med.  Tniv.  Kioto,  191S  (2),  115. 

•  Antigehitiiiase  has  also  been  obtained  by  Bertiau,  Cent.  f.  Bakt.,  1914  (74) 
374. 


AUTOLYSIS  OF  BACTERIA  113 

Other  observers  have  found  that  immunization  against  Hving  or  dead 
bacteria  leads  to  the  production  of  substances  antagonistic  to  their 
enzymes,  but  the  degree  of  resistance  acquired  is  never  great,  v.  Dun- 
gem"  found  that  the  serum  of  animals  infected  with  various  bacteria 
prevented  digestion  of  gelatin  by  the  enzymes  obtained  from  cultures 
of  the  same  species  of  bacteria.  He  applied  this  fact  to  the  diagnosis 
of  infectious  conditions,  finding  that  the  serum  of  a  patient  with 
osteomyelitis  was  over  twenty  times  as  strongly  inhibitory  to  staphy- 
lococcus enzymes  as  was  serum  of  normal  persons.  The  reaction  is 
specific,  cholera  vibrio  enzymes  not  being  inhibited  to  any  correspond- 
ing degree. 

Kantorowicz^  and  de  Waele^  state  that  bacteria  contain  an  intra- 
cellular anti-protease  which,  with  most  bacteria,  holds  in  check  the 
proteolytic  action;  only  with  the  liquefying  bacteria  are  the  proteases 
in  excess.  Bacteria  grow  well  in  strong  solutions  of  enzymes,  and  with- 
out destroying  the  enzymes  (Fermi).'"  After  Gram-negative  bacteria 
have  been  heated  to  80°  they  are  readily  digested  by  trypsin,  pepsin 
or  leucocytic  proteases;  but  Gram-positive  bacteria  are  resistant  even 
after  heating.  This  is  ascribed  by  Jobhng  and  Petersen"  to  the  un- 
saturated fatty  acids,  which  are  present  in  greater  amounts  in  Gram- 
positive  bacteria. 

Autolysis  of  Bacteria. — Autolysis  occurs  also  in  bacteria,  their  pro- 
teolytic enzymes  digesting  the  cell  substance  whenever  the  organisms 
are  killed  by  agents  (chloroform,  toluene,  etc.)  that  do  not  destroj' 
these  enzjaiies,  and  which,  being  fat  solvents,  may  facilitate  digestion 
by  removing  the  inhibitory  lipoids.  Even  the  absence  of  food  leads 
to  autolysis,  presumably  because  the  normally  existing  autolj'tic 
processes  are  not  counteracted  by  synthesis  of  new  protein  material; 
hence,  autolysis  occurs  when  bacteria  are  placed  in  salt  solution  or 
distilled  water.  Although  it  had  been  known  for  many  years  that 
yeast  cells  digest  one  another  when  there  is  nothing  else  for  them 
to  live  upon,  the  first  definite  study  of  bacterial  autolysis  seems  to  have 
been  made  by  Levy  and  Pfersdorff'^  ^nd  Conradi.'^  The  former 
digested  anthrax  bacilli  (in  whose  bodies  are  contained  rennin,  lipase 
and  protease)  under  toluene  for  several  weeks  and  obtained  a  slightly 
toxic  product.  Conradi  permitted  dj'sentery  bacilli  and  typhoid 
bacilli  to  digest  themselves  in  normal  salt  solution  for  twenty-four  to 
forty-eight  hours  at  37°  C.,  and  obtained  in  this  way  the  soluble,  highly 
poisonous  endotoxins  of  the  bacteria,  which  are  liberated  by  the  de- 
struction of  the  bacterial  structure  by  the  autolytic  enzymes.     Longer 

^  Miinch.  med.  Woch.,  1898  (45),  10-10. 

8  Miinch.  med.  Woch.,  1909  (56),  897. 

9  Cent.  f.  Bakt.,  1909  (50),  40. 

1"  Arch.  FarmacoL,  1909  (8),  481. 
"Jour.  Exp.  Med.,  1914  (20),  321. 
1-  Deut.  med.  Woch.,  1902  (28),  879. 
13  Ibid.,  1903  (29),  26. 


114  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

autolysis  results  in  the  destruction  by  the  enzymes  of  the  endotoxins 
themselves.  Rettger^^  found  among  the  autolj^ic  products  of  bac- 
teria, leucine,  tyrosine,  basic  substances,  and  phosporic  acid.  Under 
favorable  conditions  complete  autolysis  can  occur  in  Iwo  to  ten 
days. 

Brieger  and  Mayer'^  found  that  at  room  temperature  (15°  C.) 
practically  no  autolysis  occurs  with  typhoid  bacilli  in  distilled  water, 
and  the  soluble  products  thus  obtained  are  quite  non-toxic,  although 
if  injected  into  animals  they  give  rise  to  the  production  of  agglu- 
tinins and  bacteriolysins.  Bertarelli'®  has  used  the  products  of  au- 
tolysis of  cholera  vibrios  successfully  in  the  production  of  immunitj^ 
and  states  that  the  products  of  autolysis  consist  largely  of  nucleins. 

It  is  probable  that  in  every  culture  bacteria  are  constantly  being 
destroyed,  either  by  their  own  enzymes  or  by  the  proteolytic  enzymes 
of  the  other  bacteria.  Some  bacteria  are  much  more  rapidly  auto- 
lyzed  than  others,  cholera  vibrios,  colon,  typhoid,  and  dj^sentery 
bacilli  being  rapidly  digested,  while  streptococci,  staphylococci  and 
tubercle  bacilli  are  very  little  and  slowly  autolyzed.  In  general,  the 
Gram-positive  organisms  resist  autolysis  longest,  but  pneumococci 
autolyze  readil}'. 

Conradi,'^  who  has  shown  that  certain  products  of  autolysis  of  tis- 
sues are  bactericidal,  believes  that  also  in  cultures  powcrfulh'  bacteri- 
cidal substances  are  produced  through  autoh'sis  of  the  bacteria.  This 
he  thinks,  accounts  for  the  decrease  in  numbers  of  living  bacteria  that 
always  sets  in  after  a  short  period  of  growth  on  artificial  media;  but 
there  is  much  doubt  as  to  these  substances  being  of  any  considerable 
importance  in  the  body.^^  It  has  been  found  by  Turro'^  that  ex- 
tracts from  various  tissues  containing  autolytic  enzymes  can  digest 
bacterial  cells. ^^  It  is  very  possible  that  the  endotoxins  contained 
within  such  pathogenic  bacteria  as  typhoid  and  cholera  are  liberated 
through  digestion  of  the  bacteria,  either  by  autolysis  or  by  the  en- 
zymes of  the  leucocytes  and  tissues  of  the  organism  that  they  have 
infected.  These,  and  a  number  of  other  bacteria,  produce  no  soluble 
toxins  that  diffuse  from  the  cells  as  do  diphtheria  and  tetanus  toxin, 
and  it  is  difficult  to  explain  liie  toxic  effects  these  bacteria  produce 
without  assuming  that  their  iiitrnccllular  toxins  are  liberated  in  some 
such  way.  It  is  also  cjuite  probable  that  th(>  enzymes  found  in  fil- 
trates from  bacterial   cultures  arc  liberated   from  tlu>  liacterial  cells 

1^  Jour.  Mod.  Research,  1904  (13),  79. 

"  Deut.  med.  Woch.,  1904  (30),  980. 

"Cent.  f.  Hiikt.,  l'.)()r>  (3S),  5SI. 

"  Miinch.  mod.  Woohon.sclir.,  1905  (ry2)    1701. 

'8Soo  iMJkinnii,  ('out.  f.  Hiikt.,  190()  (41),  3(17;  I'assini,  AVion.  klin.  Woch., 
190<)  (19),  (127. 

'"  Cent.  f.  liukt.,  1902  (32),  10."). 

*"  Si^;wurl,  (Arl).  ii.  d.  i'liMi.  Inst.  Tiil)inK<'n,  1902  (3),  277)  found  tl.at  trypsin 
and  poi),siri  (witliout  acid)  do  not  injuro  liviufi;  antluax  bacilli. 


POISONOUS  BACTERIAL  PRODUCTS  Ho 

only  when  these  have  been  uutolyzed.-'  With  the  possibh;  exception 
just  mentioued,  there  is  httle  evidence  that  the  bacterial  enzymes 
play  any  important  role  in  infectious  diseases.  They  may  be  a  slight 
factor  in  the  digestion  of  tissue  and  exudates  in  suppuration,  but  as 
compared  with  the  leucocytic  enzymes  their  influence  is  probably  mi- 
nute; beyond  this  they  have  no  apparent  influence  upon  their  host, 
and  are  chiefly  concerned  in  the  metabolism  of  the  bacteria.  The 
proteoses  and  peptones  produced  by  bacterial  action  and  isolated 
from  cultures  do  not  seem  to  be  any  more  toxic  than  those  produced 
by  pepsin  and  trypsin,  but  violent  poisons  may  be  liberated  from 
bacteria  during  autolysis,  as  Rosenow^^  has  shown  for  the  pneumo- 
coccus  and  other  bacteria;  these  poisons  seem  similar  to  or  identical 
with  the  so-called  anaphjjlatoxin  which  is  supposedly  formed  by  the 
digestion  of  bacteria  with  serum  complement,  and  presumably  they 
are  proteoses  or  polypeptids,  but  their  exact  nature  is  not  known. 
(See  Anaphylatoxin,  Chap,  vii.) 

POISONOUS  BACTERIAL  PRODUCTS 

Almost  without  exception  all  the  harm  that  bacteria  do  is  brought 
about  by  means  of  the  chemical  substances  produced  in  one  vray  or 
another  by  their  metabolic  processes.  Animal  parasites  may  do  harm 
mechanically,  but  with  the  possible  exception  of  the  effects  of  capil- 
lary emboli  (especially  with  anthrax),  bacteria  produce  all  their  ef- 
fects through  chemical  means.  The  poisonous  chemical  substances 
produced  by  bacteria  are  commonly  grouped  into  four  classes: 

I.  Products  of  the  decomposition  of  the  media  upon  which  the 
bacteria  are  growing;  among  these  the  best  known  are  the  ptomams. 

II.  Soluble  poisons  manufactured  by  the  bacteria,  and  secreted 
from  the  cell  into  its  surrounding  media — the  true  toxins. 

III.  Poisons  manufactured  by  the  bacteria  which  do  not  escape 
from  the  normal  cell  but  which  are  as  specific  in  their  poisonous  prop- 
erties as  the  true  toxins;  because  of  their  intracellular  situation  they 
are  called  endotoxins. 

IV.  Poisonous  protein  constitutents  of  the  bacterial  cell  which 
form  part  of  the  cell  protoplasm,  but  which  are  not  soluble,  and  the 
poisonous  effects  of  which  are  not  specific  and  not  usually  responsible 
for  the  disease;  these  are  called  bacterial  proteins. 

21  Emmerich  and  Loew  (Zeitschr.  f.  Hyp.,  1899  (31),  1),  having  found  that 
pyocyanase  is  capable  of  destroying  and  digesting  other  bacteria  than  pyocy- 
aneus,  suggested  that  it  might  be  a  potent  factor  in  producing  artificial  immu- 
nity. Their  rather  remarkable  hypotheses  have  been  much  contested,  and  are 
of  questionable  value.  (See  Petrie,  Jour,  of  Pathol,  and  Bacteriol.,  1903  (8), 
200;  also,  Rettger,  Jour.  Infectious  Diseases,  1905  (2),  5G2;  Emmerich,  Miinch. 
med.  Woch.,  1907  (54),  2217). 

"  Jour.  Infect.  Dis.,  1912  (10),  113;  (11),  94,  235  and  480. 


116  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

Ptomains 

Ptomains,  the  soluble  basic  nitrogenous  substances  that  are  found 
in  the  medium  in  which  bacteria  have  been  growing,  were  the  first 
bacterial  products  that  were  recognized,  and  for  some  time  it  was 
believed  that  it  was  through  the  production  of  such  alkaloid-like  sub- 
stances that  bacteria  caused  disease,  just  as  poisonous  plants  owe 
their  effects  to  poisonous  alkaloids.  It  was  soon  found,  however,  that 
the  ptomains  that  could  be  isolated  from  cultures  of  pathogenic  bac- 
teria were  insufficient  by  themselves  to  cause  the  poisonous  effects 
that  such  cultures  produced  when  injected  into  animals.  The  isolated 
ptomains  were  not  onlj^  far  less  poisonous  than  the  original  culture, 
but  furthermore  they  did  not  produce  the  symptoms  and  anatomical 
changes  characteristic  of  the  diseases  that  the  pathogenic  organism 
caused.  Moreover,  the  majority  of  ptomains  are  not  very  poisonous, 
and  highly  poisonous  ptomains  may  be  produced  by  non-pathogenic 
bacteria.  As  a  result,  the  work  on  ptomains,  which  once  occupied 
many  laboratories  and  promised  to  reveal  the  entire  chemistrj^  of  bac- 
terial intoxication,  has  now  been  almost  completely  dropped.  The 
interest  in  ptomains  is  by  no  means  entirely  historical,  however,  for  it 
is  possible  that  poisonous  ptomains  at  times  do  enter  the  body  and 
cause  illness,  perhaps  even  death.  The  close  chemical  resemblance  to 
vegetable  alkaloids  of  some  of  the  ptomains  that  may  arise  in  decom- 
posing corpses,  makes  them  of  great  importance  to  chemists  searching 
for  the  cause  of  death  in  cases  of  supposed  poisoning.  Therefore  the 
most  essential  features  of  the  ptomains  and  their  chief  known  rela- 
tions to  intoxications  will  be  briefly  discussed,  referring  the  reader 
for  a  full  consideration  to  Vaughan  and  Novy's  "Cellular  Toxins" 
and  Barger's  "The  Simpler  Natural  Bases." 

The  ptomains  owe  their  basic  character  to  nitrogen-containing 
radicals,  principally  amino-groups,  and  hence  arc  formed  from  ni- 
trogenous substances,  chiefly  proteins,  which  contain  their  nitrogen 
in  the  amino  form.  Probably  most  ptomains  arise  from  the  decompo- 
sition of  the  protein  medium  upon  which  the  bacteria  grow,  although 
undoubtedly  part  of  the  ptomains  is  also  formed  from  the  destruc- 
tion of  the  bacterial  cells  themselves;  how  large  a  i)art  of  the  pto- 
mains is  formed  by  intracellular  bacterial  processes  and  how  much 
by  cleavage  of  the  proteins  of  the  media  by  extracellular  bacterial 
enzymes  is  unknown.  The  structure  of  the  ptomains  shows  them  to 
be  V(!ry  closely  related  to  the  amino-acids  obtained  ])y  cleavage  of  the 
prot(;in  molecule  by  enzymes  antl  other  hj'drolytic  agencies;  and  the 
determination  of  the  composition  of  the  several  amino-acids  of  the 
proteins  lias  quite  cleared  up  the  problem  of  the  origin  of  the  pto- 
mains. Presumably  these  secondary  changes  result  from  the  action 
of  special  enzynu^s  upon  th(>  aniiiio-acids.  Most  of  the  jitomains  are 
fre(!  from  or  ]nn)v  in  oxygen,  lieiicc^  rcihiction  processes,  or  lack  of  sufli- 
ciciit  oxygen  foi-  oxidation,  are  prob;il>ly  ini|)ortaiit  in  t  heir  proihiction. 


PTOMAlNS  117 

The  poisonous  ptomains,  which  are  decidedly  in  the  minority  among 
the  entire  group,  are  themselves  subject  to  decomposition,  being 
most  abundant  in  the  cultures  after  a  certain  period  of  time,  and  then 
decreasing  in  amount.  Very  old  cultures  show  almost  none  of  the 
higher  molecular  forms  of  nitrogen,  such  as  ptomains,  these  substances 
having  been  changed  into  anmionium  and  nitrate  compounds.  In 
sharp  contradistinction  to  the  toxins,  the  yiomalns  are  by  no  means 
specific.  No  matter  upon  what  medium  diphtheria  bacilli  grow,  the 
toxin  produced  has  qualitatively  the  same  properties,  whereas  the 
nature  of  the  ptomains  depends  not  only  upon  the  nature  of  the  bac- 
teria producing  them,  but  also  even  more  upon  the  sort  of  soil  upon 
which  the  bacteria  are  grown,  the  temperature,  the  duration  of  the 
process,  and  the  quantity  of  oxygen  furnished.  The  same  organism 
may  produce  totally  different  ptomains  when  grown  on  different 
media  or  under  different  conditions.  Another  essential  difference 
is  that  we  cannot  obtain  an  immune  serum,  antagonizing  the  action 
of  ptomains,  by  injecting  ptomains  into  animals. 

If  ptomains  do  cause  intoxications  presumably  it  is  when  they  are 
taken  in  with  food  in  which  they  have  been  produced  b}-  bacterial  de- 
composition. Besides  this  food  poisoning,  it  is  also  possible  that  pto- 
mains may  be  formed  by  putrefaction  within  the  gastrointestinal 
tract.  Another  possible  source  of  ptomains  is  furnished  by  decom- 
posing tissues  in  gangrene.  It  is  doubtful  if  ptomains  are  produced 
in  sufF.cient  quantities  by  pathogenic  bacteria  infecting  living  tissue 
to  be  of  any  importance.  Food  poisoning  is  by  no  means  uncommon, 
but  we  do  not  know  how  often  it  is  due  to  ptomains;  it  may  be  the 
result  of  poisonous  materials  contained  abnormally  in  the  food,  that 
are  not  ptomains,  e.  g.,  botuhsm;  or  it  may  be  due  to  an  infection  of 
the  animal  from  which  the  meat  came  with  pathogenic  organisms, 
particularly  the  B.  enteritidis  of  Gaertner  and  other  bacteria  related 
to  the  colon-t3'phoid  group;  or  in  other  ways  food  ordinarily  wholesome 
may  become  poisonous.-^  The  commonest  sources  of  ptomain  poison- 
ing are  supposed  to  be  imperfectly  preserved  canned  meats,  sausages, 
decomposing  fish,  cheese,  ice-cream,  and  milk.-^^ 

Chemical  Composition  of  Ptomains. — To  indicate  the  composition  and  nature 
of  ptomains  a  few  of  the  more  important  ones  will  be  described.  As  illustrative 
of  the  simpler  forms  may  be  mentioned: 

Methvl  amine,  CHs-NH.. 

Di-methyl  amine,  CH3  -  XH  -  CH3. 

Tri-methyl  amine,         CH3  -  N  -  CH3. 

I 
CH3. 

These  bodies,  which  are  commonly  found  in  decomposing  proteins  are  but  very 
slightly  toxic,  and  of  little  pathological  importance. 

23  See  Jordan,  "Food  Poisoning,"  University  of  Chicago  Press,  1917. 
-•'  All  these  matters  are  discussed  at  length  by  Vaughan  and  Xovy,  to    whose 
book  the  reader  is  referred. 


118  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

The  source  of  the  ptomains  in  the  various  amino-acids  is  usuall}'  easily  traced 
through  their  chemical  structure,  and  Ackermann  and  Kutscher"  have  classified 
them  in  this  relation  under  the  name  " aporrhegma." 

When  we  examine  the  structural  formulae  of  some  of  the  larger  ptomaln  mole- 
cules and  compare  them  with  the  formula"  of  the  amino-acids  that  form  the  protein 
molecule,  the  relation  is  apparent,  e.  g.,  compare  iso-amylamine  with  leucine. 
CHav  CH3\ 

^CH-CHj-CHo-NH,  ^CH-CHo-CH-XH, 

CH/  .  CH3  aeucine)      ^COOH. 

(iso-amylamine) 

Putrescine,   C4H12N2,  structural  formula, 

NH2-CH,-CH2-CH2-CH2-XH2, 
and  cadaverine,  C5H14N2,  structural  formula, 

XH2  -  CH2  -  CH2  -  CH2  -  CH2  -  CH2  -  XHo, 

are  of  interest  because  they  have  been  found  in  the  intestinal  contents,  arising  from 
putrefaction  of  proteins,  and  also  are  sometimes  present  in  the  urine  in  cystinuriaJ^ 
They  are  closely  related  to  the  diamino-acids,  lysine  and  ornithine.  Thej'  are 
but  slightly  toxic,  although  capable  of  causing  local  necrosis  Avhen  injected  sub- 
cutaneously.     (See  further  discu.ssion  on  these  and  the  Pressor  Bases  in  Chap,  xxi.) 

The  Choline  Group. — Another  group  of  ptomains,  including  cho- 
line and  closely  related  substances,  is  also  of  interest.  These  ptomains 
are: 

Chohne,  CH2OH— CH2— X(CH3)3— OH 

Xeurine,  CH2=CH— X(CH3)3— OH 

Muscarine,  CH(0H)2— CH>— X  (CH3).-r-0H 

Betaine,  COOH— CH,— X(CH3)3— OH 

The  first  point  of  importance  is  that  choline  is  present  in  every 
cell  normally,  forming  the  nitrogenous  portion  of  the  lecithin  mole- 
cule. Its  source  in  putrefaction  of  tissues  is,  therefore,  plain.  It  is 
possible  that  choline  is  liberated  from  nerve  tissues  when  they  break 
down  in  the  body  during  hfe,"  and  there  is  a  considerable  literature 
on  the  supposed  finding  of  choline  in  the  blood  and  cerebrospinal 
fluid  in  diseases  of  the  central  nervous  system  and  experimental 
lesions  in  nervous  tissues.  At  present  it  seems  probable  that  these 
observations  depend  upon  faulty  methods  of  analj'sis,  and  it  is  ex- 
tremely doubtful  if  enough  choline  is  ever  set  free  at  one  time  from 
even  severe  acute  nervous  lesions  to  be  detected  in  the  body  fluids  by 
chemical  means. ^^  Hunt^^  has  devised  a  physiological  test  that  per- 
mits of  the  detection  of  as  little  as  0.00001  mg..  but  he  was  unable  to 

"  Zeit.  physiol.  Chem.,  1910  (69),  2G5. 

2«Udrdnsky  and  Baumann,  Zeit.  physiol.  Choiii.,  1889  (13),  562;  1889  (15) 
77. 

"  Coriat  (Amer.  Jour,  of  Physiol.,  1904  (12),  353)  has  studied  the  conditions 
under  which  choline  may  be  produced  from  lecithin.  Putrefaction  of  lecithin 
or  lecithin-ridi  tissues  liberates  choline  as  also  does  aut()ly.-;is  of  brain  tissue; 
neither  pcjisin  nor  trypsin,  however,  splits  it  from  the  lecitliin.  In  brain  tissue, 
therefore;,  there  seems  to  be  an  enzyme  different  from  trypsin,  which  splits  choline 
out  of  tlie  lecithin  molecule. 

-"Sec  Wehstcr,  liioclicm.  .lour.,  1900  (4),  123 ;  Kajiura.  (.luart.  Jour.  Exper. 
Physiol..  190.S  (1),  291;  llandel.sniuiin,  Deu).  Zeit.  Nervenheilk.,  1908  (35),  428; 
Dori'c  and  (Jolla,  Biochem.  Jour.,  1910  (5),  306. 

-"Jour.    I'liurmacol.,    1915   (7),   301. 


PTOMA'iNS  119 

obtain  evidence  that  choline  is  of  any  sip;nificancc  in  either  physiologi- 
cal or  patholofj;i('al  processes.  Normally  the  largest  amounts  by  far  are 
obtained  from  the  adrenals,  which  also  seem  to  contain  choline  deriva- 
tives of  much  greater  physiological  activity.  Choline  itself  is  some- 
what toxic,  but  the  closely  related  body,  neurine,  into  which  it  may  be 
transformed,  is  highly  j)oisonous,  which  makes  chf)line  an  important 
indirect  source  of  intoxication.  It  is  possible,  for  example,  that 
lecithin  taken  in  the  food  splits  off  choline  in  the  gastro-intestinal 
tract,  and  this  being  converted  into  neurine  gives  rise  to  intoxication 
which  may  be  ascribed  to  food  intoxication.  Likewise  it  has  been 
suggested  that  the  intoxication  of  fatigue  may  be  due,  at  least  in  part, 
to  choline  and  neurine  produced  from  lecithin  decomposed  during  the 
period  of  cellular  activity.  The  close  structural  relation  to  choline 
and  neurine,  of  the  mushroom  poison,  muscarine,  which  produces 
physiological  effects  very  similar  to  those  of  neurine,  indicates  the 
close  relationship  of  the  putrefactive  ptomains  and  the  vegetable 
alkaloids.  Indeed  a  muscarine  apparently  identical  with  that  of  the 
mushroom  has  been  found  in  decomposing  flesh,  and  neurine,  presum- 
abl}^  derived  from  lecithin,  may  be  found  in  human  urine. ^"  Betaine, 
the  fourth  member  of  the  group,  which  has  but  slight  toxicity,  is 
particularly  well  known  as  a  constituent  of  plant  tissues. 

Both  neurine  and  muscarine  are  extremely  poisonous  and  quite 
similar  in  their  effects.  Subcutaneous  injection  of  but  1  to  3  mg. 
of  muscarine  in  man  produces  salivation,  rapid  pulse,  reddening  of 
the  face,  weakness,  depression,  profuse  sweating,  vomiting,  and  di- 
arrhoea. Neurine,  likewise,  causes  salivation,  lachrymation,  vomiting, 
and  diarrhoea.  In  fatal  poisoning  respiration  ceases  before  the  heart 
stops.  Both  poisons  resemble  physostigmine  in  their  stimulation  of 
secretion  and  are  equally  well  counteracted  by  atropine.  The  toxicity 
of  these  substances  is  so  great  that  not  a  large  amount  would  need 
to  be  formed  by  oxidation  of  choline  to  produce  severe  symptoms, 
although  it  is  not  known  that  this  actually  occurs  in  the  body.  When 
introduced  by  mouth,  the  lethal  dose  of  neurine  is  ten  times  as  great 
as  when  injected  subcutaneously,  indicating  that  chemical  changes  in 
the  gastro-intestinal  tract  or  liver  offer  some  protection  against  in- 
toxication by  these  substances  when  taken  in  tainted  food.  Choline, 
although  by  no  means  so  poisonous  as  neurine,  has  a  similar  action 
when  administered  in  sufficiently  large  doses.  According  to  Brieger, 
it  is  about  one-tenth  to  one-twentieth  as  toxic  as  neurine. ^^     Choline 

'"  Kutscher  and  Lohmann,  Zeit.  physiol.  Chem.,  1906  (48),  1. 

''  Halliburton,  "Chem.  of  Muscle  and  Nerve,"  1904,  p.  119,  states  that  choline 
produces  a  fall  in  blood  pressure  by  dilating  the  peripheral  vessels,  whereas  neur- 
ine constricts  the  peripheral  vessels;  he  uses  this  difference  in  phj'siological  ef- 
fect as  a  means  of  distinguishing  the  two  substances.  Injected  into  animals, 
choline  causes  a  considerable  but  transient  decrease  in  the  number  of  leucocj'tes 
in  the  blood,  followed  later  by  an  increase  (Werner  and  Lichtenberg,  Deut.  med. 
Woch.,    1906    (32),    22). 


120  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

seems  to  be  rapidlj^  destroyed  in  the  body,  not  appearing  in  the  urine^^ 
but  forming  formic  acid  and  perhaps  glyoxyhc  acid.  Donath^^ 
found  that  choline  injected  directly  into  the  cortex  or  under  the  dura 
is  extremely  toxic,  causing  severe  tonic  and  clonic  convulsions,  and 
believes  that  choline  may  be  responsible  for  epileptic  convulsions. 
This  view  has  been  opposed,  and  properly  so,  by  Handelsmann-^  and 
others.  The  attempt  to  ascribe  importance  to  choline  as  a  cause  of 
either  toxic  or  therapeutic  effect  of  x-rays  seems  also  to  be  entitled  to 
but  slight  consideration.^'*  It  is  probably  a  factor  in  the  lowering  of 
blood  pressure  which  results  from  injection  of  extracts  of  various  tis- 
sues, in  which  it  is  commonly  present  in  minute  amounts,^'  for  very 
minute  amounts  of  choline  will  produce  a  decided  fall  in  blood  pres- 
sure.^^ 

The  Pressor  Bases. — By  decarboxylation  of  amino  acids,  amines  are  obtained, 
and  some  of  them,  notablj^  those  derived  from  leucine,  tyrosine,  phem-lalanine  and 
histidine,  have  a  marked  effect  on  non-striated  muscle.  These  are  discussed  in 
Chapter  xxi. 

Toxins 

Certain  bacteria  produce  soluble  poisons  by  sj^nthetic  processes, 
which  poisons  are  secreted  into  the  surrounding  medium  and  repre- 
sent the  chief  poisonous  products  of  the  bacteria,  being  capable  of 
causing  most  or  all  of  the  symptoms  attributed  to  infection  by  the 
specific  bacteria  that  have  manufactured  them.  To  this  class  of  solu- 
ble poisons  the  term  toxin  has  now  become  limited  (for  reasons  that 
will  be  mentioned  below),  including  not  only  toxins  of  bacterial  ori- 
gin, but  also  poisons  of  similar  nature  produced  by  animals  (snake 
venoms,  eel  serum,  etc.)  and  by  plants  (ricin,  abrin,  crotin).  The 
chief  bacteria  secreting  true  toxins  are  B.  diphtherice,  B.  tetani,  B. 
pyoajaneus,  and  B.  botulinus.  Dysentery  bacilli,  the  anaerobes  of 
gas  gangrene,  and  perhaps  a  few  other  pathogens  also  secrete  a  toxin. 
Pick  considers  the  active  constituent  of  tuberculin  to  be  a  true  toxin, 
or  closely  related  thereto.  Also  the  hemoljiic  poisons  produced  by 
many  bacteria  seem  to  be  true  toxins.  It  will  be  seen  that  the  term 
toxin  has  been  greatly  narrowed  since  the  time  when  all  ptomains  and 
other  poisonous  bacterial  products  were  called  toxins,  until  now  it  has 
come  to  include  the  specific  poisons  of  but  a  few  of  the  great  group  of 
pathogenic  bacteria. 

Chemical  Properties  of  Toxins. — The  chemical  nature  of  the 
toxins  is  entirely  unknown.     By  various  precipitation  methods  they 

32  V.  Iloosslin,  Hofmeister's  Beitr.,  1906  (S),  271. 

^•' Zeit.  f.  physiol.  Chciii.,  190:5  (159),  52(5;  also  see  Med.  News,  1905  (86),  107, 
for  literature  and  incthods  of  analysis. 

^«Sce  Schenk,  Deut.  med.  Wocli.,  1910  CMt),  1130. 

"  Schwarz  and  Ledercr,  I'lliigcr's  Arch.,  1908  (124),  353;  Kinoshita,  ibid.,  1910 
(132),  607. 

•'•  Mendel  cl  al.  Jour.  I'harin.  and  Exj).  Ther.,  1912  (3),  648;  Hunt  and  Taveau. 
liulletin  73,  llyg.  Lab.  U.  H.  V.  U.  Service. 


TOXINS  121 

may  be  carried  down,  but  incliukMl  with  them  arc  masses  of  impurities, 
chiefly  proteins.  They  behave  hke  electro-positive  colloids,"  but 
diffuse  faster  than  proteins.  It  is  not  certain  that  toxins  are  not 
proteins,  for  although  certain  investigators  report  that  by  purification 
processes  very  active  toxins  have  been  obtained  that  did  not  give 
the  protein  reactions,  yet  the  toxins  are  attacked  by  proteolytic  en- 
zymes, and,  like  proteins,  are  precipitated  by  nucleic  acid  (Kossel). 
Furthermore,  accumulating  experience  with  immunological  processes 
adds  increasing  doubt  as  to  the  possibility  of  antibody  formation 
being  incited  by  anything  but  proteins.  Oppenheimer  says  of  the 
toxins,  "we  must  be  contented  to  assume  that  they  are  large  mo- 
lecular complexes,  probably  related  to  the  proteins,  corresponding  to 
them  in  certain  properties,  but  standing  even  nearer  to  the  equally 
mysterious  enzymes  with  whose  properties  they  show  the  most  ex- 
tended analogies  both  in  their  reactions  and  in  their  activities."  These 
similarities  between  toxins  and  enzymes  are  very  striking,  and  in 
discussing  the  nature  of  the  enzymes  we  have  mentioned  the  reasons 
for  considering  them  related  to  the  toxins;  we  may  now^  take  up  the 
other  side  of  the  question  and  consider  the  relation  of  the  toxins 
to  the  enzymes. 

Resemblance  to  Enzymes. — First  of  all  w^e  meet  the  same  diffi- 
culty in  isolating  toxins  that  we  do  in  isolating  enzymes.  "A  pure 
toxin  is  as  unknown  as  a  pure  enzyme"  (Oppenheimer).  At  first 
both  were  believed  to  be  proteins;  now  both  are  considered  by  many 
not  to  be  proteins,  but  molecular  complexes  of  nearly  equally  great 
dimensions.  That  toxins,  like  enzymes,  are  colloids,  has  been  abun- 
dantly demonstrated.^*  Both  pass  through  porcelain  filters,  but  both 
lose  much  of  their  strength  in  the  process,  and  they  are  almost  en- 
tirely held  back  by  dialyzing  membranes.  They  behave  similarly  as 
regards  adsorption  by  suspensions,^^  and  have  similar  effects  on  the 
physical  properties  of  their  solutions  (Zunz)."*"  Neither  will  with- 
stand boiling,  and  most  forms  are  destroyed  at  80°  instantly  or  in 
a  very  short  time;  on  the  whole,  however,  toxins  are  more  susceptible 
to  heat,  as  well  as  to  most  other  injurious  agencies.  Both  stand  dry 
heat  over  100°,  and  extremely  low  temperature,  without  much  injury. 
Left  standing  in  solution  for  some  time  they  gradually  lose  their 
specific  properties,  and  in  each  case  this  seems  to  be  due  to  an  altera- 
tion in  the  portion  of  the  molecule  that  produces  the  destructive 
effects  {toxophore  or  zymophore  group),  while  the  portion  of  the  mole- 
cule that  unites  with  the  substance  that  is  to  be  attacked  (haptophore 
group)  remains  uninjured,  the  toxin  becoming  a  toxoid,  the  enzyme 

"  Field  and  Teague,  Jour.  Exper.  Med.,  1907  (9),  86. 

'8  See  Zangger,  Cent.  f.  Bakt.  (ref.),  1905  (36),  239. 

'^  By  fiocculation  of  the  colloids  bearing  adsorbed  toxins  it  may  be  possible 
to  secure  them  in  comparatively  pure  condition  (London,  Compt.  Rend.  Soc. 
Biol.,  1917  (80),  756. 

"Arch,  di  Fisiol.,  1909  (7),  137. 


122  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

a  fermentoid.  Enzymes  as  well  as  toxins  are  poisonous  when  injected 
into  animals,  and  the  animals  react  to  each  by  producing  substances 
(antibodies)  that  render  each  inert,  probably  in  the  same  way.  On 
the  other  hand,  enzymes  and  toxins  seem  to  produce  their  effects  ac- 
cording to  different  laws: — A  small  amount  of  enzyme  can  in  course 
of  time  produce  an  almost  indefinite  amount  of  effect,  whereas  toxins 
act  more  nearly  quantitatively.  It  seems  as  if  the  enzyme  were  bound 
to  the  body  upon  which  it  acts,  as  is  the  toxin,  but  that  after  it  has 
destroyed  this  body  it  is  set  free  in  a  still  active  form,  ready  to  accom- 
plish further  work,  whereas  the  toxin  is  either  not  set  free,  or  it  be- 
comes inactive  after  it  has  once  bepn  combined. 

Agencies  Destroying  or  Modifying  Toxins. — Toxins  are  very  sus- 
ceptible to  light,  direct  sunlight  soon  destroying  the  power  of  toxin 
solutions.  Fluorescent  substances  destroy  toxins  both  in  vitro  and 
in  the  body.'*^  They  are  generally  destroyed  by  moist  heat  of  80°,  but 
resist  100°  when  dry.  Oxygen,  even  dilute  as  in  air,  is  harmful;  and  all 
oxidizing  agents,  including  oxidizing  enzymes,  destroy  them  quicld3^■*- 
Like  enzymes,  they  withstand  such  antiseptics  as  chloroform,  tol- 
uene, etc.,  and  are  precipitated  by  the  heavy  metals.  Some  agencies 
seem  to  attack  only  the  toxophore  portion  of  the  molecule,  e.  g.,  iodin, 
carbon  disulphid  (Ehrlich).  Certain  toxins  (diphtheria,  dysentery) 
can  be  converted  into  non-toxic  modifications  by  acids,  the  original 
toxicity  being  restored  by  bases  (Docrr),''^  which  fact,  Pick  maintains, 
is  in  support  of  the  protein  nature  of  toxins.  Salts  of  monovalent 
metals  have  no  effect  on  toxins,  but  bivalent  and  trivalent  salts  are 
injurious  to  them,  tetanus  toxin  being  more  sensitive  than  diphtheria 
toxin.     X-rays  are  said  to  weaken  them.^'* 

'Introduced  into  the  gastro-intestinal  tract,  most  bacterial  toxins 
are  not  absorbed  (botulinus  toxin  excepted),  cause  no  symptoms,  and 
do  not  reappear  in  the  feces;  they  are  therefore  destroyed  by  the 
contents  of  the  tract,  pepsin,  pancreatic  juice,  and  bile  all  being  capa- 
ble of  destroying  toxins. ^^  They  maj%  however,  when  injected  sub- 
cutaneously,  circulate  unimpaired  in  the  blood  of  non-susceptible 
animals,  gradually  disappearing,  more  through  slow  processes  of  de- 
struction than  by  elimination.  When  injected  into  susceptible  animals, 
they  soon  disappear  from  the  blood,  being  fixed  in  the  organs  that 
they  attack.  Toxins  are  also  bound  ])y  lipoids,  fats  and  similar 
substances,  which  accounts,  at  least  in  part,  for  the  affinity  of  tetanus 

^'  Literature  Kiven  by  Not^uchi,  Jour.  Kxppr.  Med.,  1900  (S),  263. 

**  According!;  to  Pitini  (liiocheni.  Zeit.,  11)10  (2.')),  257)  toxins  cause  their  harm- 
ful effcot.s  1)V  reihiciiiK  tlie  oxidizing  capacity  of  the  tissues. 

<' Wien.  klin.  Wocli.,   1907  (20),  T). 

<'  (lerlKirtz,   Herl.  klin.   Wocli.,   1909  (40),   1800. 

•"'  IJuldwin  and  Levone  (.lour.  Mi-d.  lie.soarch,  1901  (()),  120)  found  that  diph- 
tlioria  ami  tetanus  toxin  are  l)otli  destroyed,  ai)parently  througli  digestion,  by 
pci)sin,  trypsin  and  jiajiain  acting  for  several  days.  Ueview  of  Literature  by 
Lust,  Ifofnieister's  lieitr.,  1901  (ii),  132.  See  Vincent,  Ann.  Inst.  Pasteur,  1908 
(22),  341. 


TOXINS  123 

toxin  for  nervous  tissues.""'  In  comnion  witli  other  colloids  they  are 
adsorbed  by  surfaces,  such  as  charcoal,  kaolin,  etc.;  such  ad.sorption 
is  accompanied  by  little  change  in  any  of  the  physical  properties  of  the 
solution,  except  an  increase  in  surface  tension  (Zunz). 

Differences  from  Ptomains. — While  ptomains  are  formed  by  cleavage  processes 
from  the  medium  upon  which  the  bacteria  grow,  and  the  same  ptomains  can  be 
produced  by  several  different  kinds  of  bacteria,  the  toxins  are  synthetic  'products 
of  absolutely  specific  iiaturc.  However,  the  toxins  seem  to  be  produced  little  if  at 
all  by  growing  the  bacteria  on  Uschinsky's  or  similar  media,  which  contain  no 
proteins,  carbohydrates,  or  fats,  but  merely  simple  organic  and  inorganic  salts 
of  known  composition.^'  Nevertheless  diphtheria  toxin  is  essentially  the  same 
no  matter  on  what  sort  of  medium  the  l)acteria  are  grown,  whereas  ptomains  vary 
with  the  nature  of  the  siilistance  from  wliich  they  are  produced.  Toxins  arc  true 
Sgcretions  of  bacterial  cells,  just  as  trypsin  is  of  pancreat'c  c.pIIs,  or  tli,Y.roiodin  of 
thyroid  cells.  SvEi-bodies  caiTTKf  produced  agamsttoxins,  but  not  against 
ptomains.  — ' ^ "  ~ 

Ehrlich's  Conception  of  the  Nature  of  Toxins. — Chemical 
studies  of  toxins  being  impossible,  we  have  been  obliged  to  study  them 
through  their  physiological  effects,  just  as  we  have  obtained  informa- 
tion concerning  enzymes  through  their  specific  actions.  In  this  way 
Ehrlich  developed  well-crj'stallized  ideas  concerning  the  structure 
of  toxins,  as  well  as  the  manner  in  which  they  act,  which  may  be  briefly 
summarized  as  follows:  Each  toxin  molecule  consists  of  a  large  num- 
ber of  organic  complexes,  grouped,  as  in  other  organic  compounds, 
as  side-chains  about  a  central  chain  or  radical.  One  or  more  of  these 
complexes  has  a  chemical  affinity  for  certain  chemical  constituents  of 
the  tissues  of  susceptible  animals,  with  which  the  toxin  molecule 
unites;  this  binding  group  is  called  the  haptophore  (meaning  "bear- 
ing a  bond").  Another  side-chain  or  group  of  side-chains  exerts  the 
injurious  effects  upon  the  tissue  to  which  the  molecule  has  been  bound 
by  the  haptophore,  and  cannot  produce  these  injurious  effects  unless 
it  has  been  so  bound.  This  injury-working  group  is  called  the  toxo- 
pJiore.  An  animal  is  susceptible  to  a  toxin  only  when  its  cells  con- 
tain substances  which  possess  a  chemical  aflB.nity  for  the  haptophore 
groups  of  the  toxin,  and  also  substances  which  can  be  harmfully  influ- 
enced b}^  the  toxophore  groups.  Tetanus  toxin,  for  example,  owes  its 
effects  to  the  fact  that  nervous  tissues  contain  chemical  substances 
having  a  strong  affinit}'  for  the  haptophore  group  of  tetanus  toxin, 
and  also  substances  that  can  be  attacked  with  serious  results  by  the 
toxophore  group  of  the  toxin.  The  nature  of  the  changes  brought 
about  by  the  toxophore  groups  of  toxins  is  not  understood;  there  are 
many  resemblances  to  the  action  of  enzymes,  but  the  analogy  is  by 
no  means  complete.  We  find  perhaps  the  closest  analogy  to  the  en- 
zymes in  the  toxic  substances  that  destroy-  red  corpuscles  and  bacteria 
(hemolysins,  bacteriolysins) ,  which  will  be  considered  in  another  place. 
The  immunity  against  toxins  and  enzymes  seems  to  be  produced  by 

^«Loewe,  Biochem.  Zeit.,  1911  (33),  225,  and  (34),  495. 
"See  Rettgerand  Robinson,  Jour.  Med.  Res.,  1917  (38),  357. 


124  CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

identical  processes,  which  consist  in  an  overproduction  of  the  cellular 
constituents  (receptors)  which  bind  the  haptophore  groups  to  the  cells, 
these  excessive  receptors  being  secreted  into  the  blood,  where  they 
combine  with  the  toxin  or  enzyme  so  that  it  cannot  enter  into  combi- 
nation" with  the  cells.  This  "side  chain  theory"  of  Ehrhch  has  been  a 
useful  working  hypothesis,  although  it  is  becoming  highly  probable 
that  it  does  not  picture  the  exact  method  of  toxin  and  antitoxin  action. ^^ 
Immune  substances  cannot  be  produced  against  ptoma'ins,  or  for 
that  matter  against  the  vegetable  alkaloids,  or  against  any  chemical 
bodies  of  known  constitution.  Another  difference  between  the  action 
of  toxins  and  simpler  chemical  poisons  is,  that  while  with  the  latter  the 
effects  are  produced  in  a  very  short  time  after  injection,  there  is  a 
latent  period  of  several  hours  before  symptoms  appear  after  injecting 
toxins.  What  occurs  during  this  latent  period  is  not  fully  known, 
but  that  there  is  a  latent  period  suggests  a  resemblance  to  enzyme 
action.  An  alkaloidal  or  other  chemical  poison  enters  the  cell,  and  its 
harm  is  done  at  once.  A  toxin  combines  with  the  cell,  and  then,  if 
it  produces  its  effects  by  an  enz3'matic  alteration  of  the  cellular  struc- 
ture, some  time  must  elapse  before  the  changes  are  great  enough  to 
cause  the  appearance  of  symptoms. 

Endotoxins" 

By 'far  the  greater  number  of  pathogenic  bacteria  do  not  secrete  their  poisons 
as  toxins  into  the  surrounding  medium,  aUhough  they  manifestly  cause  disease  by 
poisoning  their  host.  Among  them  are  such  organisms  as  the  typhoid  bacilhis. 
pneumococcus,  the  pus  cocci,  cholera  vibrios,  and  many  others.  If  cultures  of 
these  organisms  are  filtered,  the  filtrate  will  be  found  to  be  but  slightly  toxic 
(except  for  the  hemolytic  i)oisons).  although  the  bodies  of  the  bacteria  after  they 
have  been  killed  by  chloroform  or  other  antiseptics  are  highly  poisonous  if  injected 
into  an  animal.  These  bacteria,  then,  produce  poisons  which  do  not  escape  from 
the  cells  into  the  culture-medium,  but  are  firndy  held  within  them.  By  using 
various  means  these  intracellular  toxins,  or  endotoxins,  can  be  obtained  independent 
of  the  bacterial  cells.  One  of  these  is  to  grind  up  the  cells,  which  can  be  particu- 
larly well  done  if  they  are  first  made  brittle  by  freezing  at  the  temperature  of 
liquid  air  (MacFadyen's  method).  By  very  great  ])ressure  in  the  Buchner  press 
the  cellular  contents  can  he  expressed.  They  may  also  be  obtainctl  by  letting  the 
bacteria  autolyze  themselves  for  a  short  time  in  non-nutrient  fluids  (Conradi,^" 
et  ai).  Endotoxins  obtained  in  this  way  are  soluble  and  highly  poisonous,  and  it  is 
undoubtedly  through  their  action  that  the  characteristic  diseases  are  produced  by 
the  bacteria  that  contain  them.  Presumably  the  endotoxins  are  liberated  in  the 
body  either  by  autolysis,  or  by  heterolysis  by  the  enzymes  of  the  body  cells  and 
fluids,  and  there  is  some  (luestion  as  to  whether  they  are  preformed  specific  con- 
stituents of  the  bacteria,  or  merely  the  jjoisonous  ])roduct  of  enzymatic  disintegra- 
tion of  the  ba(!terial  i)roteins,  similar  to  the  "anaphylatoxins."^' 

Endotoxins  differ  from  the  true  toxins  in  one  imj)ortant  respect:  namely,  it  is 
difficuU    or  impossible  to  obtain  an  antitoxin  for  endotoxins  by  immunization  of 

<«  See  Coca,  .lour.  Infect.  Dis.,  1915  (17),  ;J51. 

*•  See  general  r(>view  by  I'feifTer,  Jahresber.  d.  Immunitatsforsch.,  1910  (ti),  13. 
"0  Deut.  med.  Woch.,  \\)0A  (29),  2ti. 

"See  Dold  and  llanau,  Zeit.  Immunitat.,  1913  (19),  31;  Zinsser,  "Infection 
and  Resistance,"  N.  W,  1911,  ("Iim]).  xvii. 


POISONOUS  BACTERIAL  PROTEINS  125 

animals.''-  Animals  immunized  against  endotoxins  develop  in  their  serum  sub- 
stances that  arc  bactericidal  and  agglutinative  to  the  bacteria  from  which  the 
poisons  are  derived,  but  the  serum  will  not  neutralize  the  endotoxins.  As  a  re- 
sult, we  are  unable  to  perform  experiments  indicating  whether  endotoxins  have 
the  same  structure  as  the  true  toxins,  i.  e.,  a  haptophore  and  a  toxophore  group, 
but  presumablj-  their  nature  is  different  in  some  essential  particular.  The  chemical 
nature  of  the  endotoxins  is  also  unknown,  for  they  are  always  obtained  mixed  with 
the  other  constituents  of  the  bacteria.*' 

Tuberculin,  once  supposed  to  be  an  albumose,  is  produced  even  when  the  bacilli 
are  grown  on  a  protein-free  medium,  and  in  the  active  solution  no  albumose  or 
other  protein  is  then  found.  Hence  it  seems  probable  that  tuberculin  is  of  the 
nature  of  a  polypeptid,  which  gives  no  biuret  reaction  but  fs  destroyed  by  pepsin 
and  trypsin,  according  to  Loevenstein  and  Pick,*^  but  not  by  erepsin  (Pfeiffer).** 
Whether  tuoerculin  should  be  considered  an  endotoxin  liberated  by  the  disintegra- 
tion of  the  bacilli  in  the  cultures  is  unknown;  Pick  looks  upon  it  as  a  secretion  of 
the  bacilli,  and  closely  related  to  the  true  toxins. 

Since  far  more  bacterial  diseases  are  brought  about  by  endotoxins  than  by  true 
toxins,  the  failure  to  secure  antitoxins  for  these  substances  has  been  a  great  check 
in  the  progress  of  serum  therapj',  and  the  problem  of  the  endotoxins  is  one  of  the 
most  important  in  the  entire  field  of  immunity. 

Poisonous  Bacterial  Proteins 

If  we  filter  a  Iwuillon  culture  of  diphtheria  bacilli  through  porcelain,  wash 
thoroughly  with  salt  solution  the  bacteria  remaining,  and  collect  them  thus  freed 
from  their  secretion  products,  it  will  be  found  that  extracts  of  the  bacterial  subtance 
or  the  bodies  of  the  killed  bacteria  themselves  are  quite  free  from  the  typical  toxin. 
This  indicates  that  the  toxin  is  eliminated  from  the  bacteria  as  fast  as  it  is  formed, 
and  no  considerable  quantity  is  retained  within  the  cell.  The  bacterial  substance, 
however,  or  proteins  isolated  from  it,  is  found  to  produce  severe  local  changes 
when  injected  into  the  bodies  of  animals,  necrosis  and  a  strong  inflammatory  reac- 
tion with  pus-formation  being  the  chief  features.  This  local  effect  is  not  a  specific 
property  of  the  diphtheria  bacillus,  for  other  bacterial  proteins,  including  proteins 
from  non-pathogenic  bacteria,  will  produce  the  same  changes;  indeed,  many 
proteins  that  are  derived  from  vegetable  and  animal  sources  have  equally  marked 
pyogenic  properties.  All  foreign  proteins  when  introduced  into  the  circulation  of 
animals  are  more  or  less  toxic,  and  the  toxic  effects  of  the  bacterial  proteins  are, 
for  the  most  part,  neither  specific  nor  particularly  striking.  There  are  a  few 
pathogenic  organisms,  however,  which  seem  to  produce  neither  true  toxins  nor 
endotoxins,  notably  the  tubercle  bacillus  and  the  anthrax  bacillus,  and  with  these 
there  may  be  a  relation  between  their  protein  constituents  and  their  specific  effects. 

Numerous  protein  substances  have  been  extracted  from  bacterial  cells,  partic- 
ularly nucleoproteins,  but  also  proteins  resembling  albvmiins,  nucleo-albumin,  and 
globulins.  In  all  probability  the  chief  proteins  of  the  bacterial  cell  are  nuclein 
compounds,  which  is  indicated  both  by  their  nuclear  staining  and  by  the  anah'ses 

"  Positive  results  are  claimed  by  Besredka  (Ann.  Inst.  Pasteur,  1906  (20), 
304),  and  some  others;  see  Kraus,  Wien.  klin.  Woch.,  1906  (19),  655;  Zeit.  Im- 
munitiit.,  1909  (3),  6-16.  It  is  suggested  by  Wasisermann  (KoUe  and  Wasser- 
mann's  Handbuch,  1912  (2),  246)  that  this  difficulty  in  obtaining  antiendotoxins 
depends  on  the  large  size  of  the  molecule, — the  small  diffusible  toxin  molecule  is 
so  altered  in  its  physical  condition  through  union  with  the  antibody  that  its 
properties  are  much  altered,  whereas  the  large  endotoxin  molecule  must  be  di- 
gested by  complement  before  its  toxicitj-  is  destroyed. 

*^  The  Aggressins  of  Bail,  to  which  he  ascribes  the  pathogenicity  of  bacteria, 
are  too  little  established  to  permit  of  a  discussion  from  the  chemical  standpoint. 
By  many  they  are  believed  to  be  nothing  more  than  endotoxins.  (Literature 
given  by  Miiller,  Oppenheimer's  Handb.  d.  Biochem.,  1909  (II  (1)  ),  681;  Dud- 
geon, Lancet,  1912  (182),  1673).     According  to  Ingravelle   (Ann.  d'  ig.  sperim., 

1910  (20),  483),  tvphoid  aggressins  are  found  in  the  albumins. 
5^  Biochem.   Zeit.,    1911    (31),   142. 

"  Wien.  khn.  Woch.,  1911  (24),  1115;  see  also  Lockmann,  Zeit.  phvsiol.  Chem., 

1911  (73),  389. 


126         CHEMISTRY  OF  BACTERIA  AND  THEIR  PRODUCTS 

of  Iwanoff;^^  and  many  of  the  nucleoproteins,  both  of  bacterial  and  non-bacterial 
origin,  cause  considerable  local  inflammatory  reaction  when  injected  into  animals. 
Tiberti'''  claims  that  vaccination  with  non-lethal  doses  of  the  nucleoproteins  of 
anthrax  bacilli  will  protect  animals  against  inoculations  of  virulent  anthrax 
bacilli.  Some  of  the  earlier  observations  on  the  toxicity  of  bacterial  proteins 
were  erroneous  because  impure  proteins,  containing  toxins,  endo-toxins,  and 
ptomains  were  used.  Schittenhelm  and  Wcichardt*^  have  found,  however, 
that  bacterial  protein.s  are  much  more  toxic  than  any  ordinaiy  proteins,  as  indicated 
by  loss  of  nitrogen,  temperature  changes  and  alterations  in  the  leucocytes  of  injected 
animals.  Furthermore,  there  are  few  other  proteins  that  produce  so  much 
inflammatory  reaction  as  the  bacterial  proteins. 

\'aughan  and  his  students  have  been  able  to  split  off  from  the  bodies  of  various 
pathogenic  bacteria  toxic  materials  which  are  stated  to  resemble  in  some  respects 
the  protamins,''^  a  though  they  do  not  all  give  a  satisfactory  biuret  test.  These 
toxic  materials  are  evidently  quite  different  from  either  the  true  soluble  toxins 
or  the  endotoxins,  since  they  resist  heating  for  ten  minutes,  at  110"^  in  the  autoclave 
with  1  per  cent,  sulphuric  acid,  this  being  a  method  used  for  securing  the  substance. 
Since  the  sarcime  and  B.  prodigionus  also  yield  similar  toxic  products,  t;hey  cannot 
be  considered  as  the  specific  toxic  substances  of  the  pathogenic  bacteria,  but  ap- 
parently are  common  to  all  proteins  of  whatever  origin.  With  .-ome  bacteria  the 
splitting  process  with  sulphuric  acid  sei)arates  completely  the  toxic  from  the  non- 
toxic insoluble  bacterial  substance,""  e.  </.,  B.  coi  communis:  with  others  a  to.xic 
portion  remains  in-:oluble.  The  colon  l)acillus  protein  gives  all  the  protein  reac- 
tions, is  synthesized  on  I'schinsky's  medium,  and  does  not  yield  a  reducing  carbo- 
hydrate. From  B.  typhosus  about  10  per  cent,  by  weight  of  protein  can  he  split 
off  b}''  dilute  acid,  of  which  at  least  a  part  seems  to  be  a  phosphorized  glyco- 
protein.*' Poisonous  substances  have  also  been  obtained  trom  B.  diphlherice, 
B.  anthracis,  B.  tuber cu'osis^^  and  B.  pyocyaneus.  They  produce  death  without 
the  usual  latent  period  observed  with  toxins,  and  are  very  toxic,  a  few  (10-20) 
milligrams  of  colon  bacillus  poison  killing  guinea-pigs  in  less  than  ten  minutes.*^ 
A  certain  degree  of  immunity  can  be  obtained  against  them.*''  Their  relation  to 
endotoxins  and  anaphylatoxins  has  yet  to  be  determined. 

Bacterial  Pigments*'' 

The  formation  of  pigment  by  bacteria  seems  to  be,  for  the  most 
part  an  adventitious,  unessential  property.  There  are  a  few  bacteria 
which  possess  pigments  of  the  nature  of  chlorophyll,  or  allied  to  it, 
and  this  pigment  is  undoubtedly  of  great  importance  in  the  life  pro- 
cesses of  these  particular  forms.  Other  varieties  of  pigment-forming 
bacteria,  of  which  but  very  few  are  pathogenic  {Bacillus  yyocijaneus, 
B.  proteus  jiuorescens,  S.  pijogenes  aureus  and  citreus,  M.  cercus 
flavus),  seem  to  produce  pigment  as  a  waste  product  which  is  excreted 
from  the  cell  as  fast  as  formed.  Generally  the  pigments  are  produced 
in  a  colorless  form  (leuco-base)  which  is  oxidized  by  the  air  into  the 
pigment,  e.  g.,  in  pyocyaneus  infections  the  soiled  dressings  are  most 

"  Hofmeister's  Beitr.,  1902  (1),  524. 

"  Cent.  f.  Bakt.,  1901)  (10),  742. 

'«  Miinch.  med.  Woch.,  1911  (58),  S41. 

'"A  full  review  of  tliis  work  is  given  in  Xauglian's  "Protein  Split  Products," 
Philadelijliin,  19i:5;  antl  in  .lour.  Lab.  Clin.  IMed.,  1911),  Vols.  1  and  2. 

«"  Wlieeler,  Jour.  Amer.  Med.,  Assoc,  1905  (44),  1271. 

«'  Ihid.,  1901  (12),  1000. 

•2  Sec  White  and  Averv,  Jour.  Med.  Res.,  1912  (20),  317. 

"'  Jour.  Amer.  Med.  .Vs.soc,  1905  (44),  1340;  American  Medicine,  1905  (10),  145. 

'*  Vauglian  (Jr.),  Jour,  of  Med.  I{esearch,  190')  (14),  ()7. 

"For  coinplele  bibliography  and  resume  see  Sullivan,  .lour.  Med.  Research, 
1905  (14),  109. 


BACTERIAL  PIGMENTS  127 

colored  about  the  portions  most  exposed  to  air.  Since  pigment-form- 
ing bacteria  produce  pigments  only  under  certain  conditions,  and  can 
grow  abundantly  without  producing  any  pigment,  it  is  evident  that 
the  pigment  formation  is  no  very  essential  part  of  their  metabolism. 
It  is  possible  to  modify  pigment  production  almost  at  will,  and  even 
to  develop  races  of  bacteria  that  do  not  produce  pigment  at  all  from 
races  that  ordinarily  are  pigment-producers. 

Of  numerous  classifications  of  pigment-forming  bacteria,  all  faulty 
because  of  our  slight  knowledge  of  the  chemistry  of  the  process,  that 
of  Migula  seems  the  best;  it  is  based  on  the  solubility  of  the  pigments 
formed,  as  follows: 

(1)  Pigments  Soluble  in  Water. — This  includes  the  pigm-ents  of  all  fluorescent 
bacteria,  as  well  as  those  giving  a  red  or  brown  color  to  gelatin  media.  Most 
important  among  these  is  Bacillus  -pyocyane^is,  whose  pigments  have  been  consider- 
ably studied.  There  seem  to  be  two  pigments,  one,  pyocyanin,  characteristic  for 
this  organism ;  and  a  fluorescent  pigment  which  numerous  other  organisms  also  pro- 
duce. Pyocyanin  has  been  analyzed  by  Ledderhose,  who  found  it  to  be  a  ptomain- 
like  body,  a  derivative  of  the  aromatic  series,  probabh'^  related  to  the  anthracenes. 
It  can  be  reduced  to  a  colorless  leuco-base,  in  which  form  it  is  probably  produced  by 
the  bacteria,  and  then  is  oxidized  in  the  air  into  the  pigment.  Its  composition  is 
ChHhN20  (the  sulphur-containing  pyocj'anin  which  has  been  described  is  probably 
impure).^*  The  fluorescent  pigment  is  insoluble  in  alcohol  and  chloroform,  and 
can  thus  be  separated  from  pyocyanin,  which  is  soluble  in  chloroform.  Although 
related  to  the  ptomains,  pyocyanin  seems  to  be  altogether  non-poisonous  to  ani- 
mals. 

Jordan^^  and  Sullivan""  have  studied  the  conditions  under  which  pigments  are 
formed,  and  found  that  pvocyauin  can  he  produced  in  protein-froe  media,  and 
without  the  presence  of  either  phosphates  or  sulphates;  but  both  sulphur  and 
phosphorus  must  be  present  to  produce  the  fluorescent  pigment.  As  pigments 
can  be  produced  on  media  containing  only  ammonium  salts  of  succinic,  lactic,  or 
aspartic  acid,  or  asparagin,  they  are  evidentlj^  formed  synthetically,  and  not  by 
cleavage  of  the  media. 

(2)  Pigments  Soluble  in  Alcohol  and  Insoluble  in  Water. — The  most  important 
bacteria  of  this  group  are  the  Slaphylococcus  pyogenes  aureus  and  citreus.  Their 
pigment  is  of  a  fatty  nature,  a  lipochrome,  which  lies  among  thebacteria  in  the  form 
of  dendritic  crystals.  Being  a  fat,  it  can  be  saponified,  and  when  decomposed  it 
gives  the  acrolein  reactions  and  odor,  from  the  breaking  down  of  the  glycerol 
of  the  fat  molecule.  Acted  upon  by  strong  sulphuric  acid,  the  yeUow  pigment 
changes  into  blue  granules  and  crystals  {iipocyanin  reaction).  The  lipochromes 
are  soluble  in  the  usual  fat  solvents,  and  form  fat  spots  on  paper. 

(3)  Pigments  Insoluble  in  Water  and  in  Alcohol. — The  pigment  of  Micrococcus 
cereus  flavus  belongs  to  this  class;  its  nature  is  quite  unknown. 

"*  Analysis  of  pyocj'anin-HCl  by  Madinaveitia  (Anales  soc.  espan.  fis.  quim., 
1916  (14),  26.3)  gave  CsoHBsNiuCioOs,  but  the  phj-sical  properties  indicated  a 
lower  molecular  weight. 

«'  Jour.  Exper.  Med.,  1899  (4),  627. 


CHAPTER  V 
CHEMISTRY   OF  THE  ANIMAL  PARASITES^ 

This  subject  has  received  much  less  consideration  than  its  import- 
ance deserves,  and  we  are  quite  in  the  dark  as  to  how  much  of  the 
effects  produced  by  animal  parasites  are  not  merely  mechanical,  but 
are  due  to  soluble  poisons  that  they  maj^  secrete  or  excrete.  Some  of 
the  parasites  probably  cause  harm  mechanically  and  in  no  other  way, 
but  with  most  of  them  there  is  more  or  less  evidence  of  the  forma- 
tion of  poisonous  substances.  The  composition  of  the  bodies  of  the 
animal  parasites  is  an  almost  unexplored  field,  but  we  have  no  reason 
to  believe  that  the  composition  of  the  cells  of  invertebrates  differs 
essentially  from  that  of  the  cells  of  higher  organisms.  Perhaps  the 
most  characteristic  constituent  observed  in  many  forms  is  chitin,  which 
forms  a  large  part  of  the  outer  covering  of  the  encysted  forms,  and 
probably  of  many  of  the  worms.  Glycogen  is  usually  abundant  in 
the  invertebrates,  and  the  animal  parasites  form  no  exception, ^  this 
carbohydrate  having  been  found  in  their  bodies  bj^  many  observers. 

Eosinophilia. — One  of  the  most  characteristic  features  of  the  animal  parasites 
is  that  they  exert  a  positive  chemotaxis  for  eosinophile  leucocytes.^ 

An  increase  in  the  number  of  these  cells  in  the  blood,  as  well  as  a  local  accum- 
ulation in  the  tissues  nearest  the  parasite,  has  been  observed  in  infection  with  prac- 
tically all  the  animal  parasites.''  Of  these,  infection  with  TrichineUa  spiralis 
causes  the  most  pronounced  eosmophilia,  presumably  because  of  the  great  number 
of  parasites  present  in  the  tissues  at  once.  That  the  eosinophilia  is  due  to  the 
action  of  the  soluble  products  or  constituents  of  the  parasites  has  been  shown  by 
experimental  injection  into  animals  of  extracts  from  the  bodies  of  the  parasites. 
Calarnida  has  found  that  extracts  of  do^  tapeworms  also,  when  placed  in  the  tissues 
in  a  capillary  tube,  cause  an  accumulation  of  eosinophile  cells  in  the  tube.^ 
Experimental  infection  with  excessive  numbers  of  trichinclla  causes  a  rapid  diminu- 
tion in  the  number  of  eosinoi)hile  leucocytes,  which  also  show  evidences  of  disinteg- 
ration in  the  bone-marrow  and  lyin])h-glands.  Such  large  injections  are  fatal, 
which  suggests  that  the  eosinoiihilia  has  a  jirotective  influence.  In  favor  of  this 
view  is  the  observation  of  Milian,"  who  foiunl  that  sarcosporidia  in  l)cef  are  des- 
troyed bj^  a  violent  leucocytic  reaction,  the  prevailing  cell  be'ng  the  eosinoi^hile. 
As  the  eosinoi)hUe  increase  does  not  occur  until  several  days  after  the  infected 
flesh  is  eaten,  the  chemotactic  substance  is  not  lilterated  from  the  encapsulated 

1  General  references  to  this  subject  will  be  found  in  v.  Fiirth's  '' Vergleichende 
chemische  Physiologic  der  nioderen  Tiere,"  .Icna,  1903;  Faust's  ''Tierische  Gifte," 
Braunschweig,  1900;  Koch,  Krgebnisse  Pathol..  1910  (XIV(l)),  41. 

2 See  Pfliiger,  Pfliigers  Arch.,  1903  (9{)),  1.'d3. 

■'Literature  by  Opie.  Amer.  Jour.  Med.  Sci.,  1904  (127),  477;  Stiiubli,  Deut. 
Arch.  klin.  Med.,  1900  (85),  280;  Iliibner,  ibid.,  1911  (104),  280;  Schwarz,  Ergcb. 
allg.  I'athol.,  1914  (17,),  138. 

'  LiteratiM-e  by  Bruns,  Liefmann  and  Miickel,  IMiincli.  med.  Woch.,  1905  (52), 
253;  Vallillo,  Arcli.  wiss.  u.  prakt.  Tierhk.,  1908  (34),  505. 

'  Negative  rcsult.s  were  olUnincd  with  extracts  of  Sclcrosloma  equinum  by 
Gro.s.so  (Folia  Ileinatol.,  1912  (11),  IS). 

•  Hull,  et  Mem.  Soc.  Anat.,  1901  (.Sor.  (i,  T.  3),  323. 

128 


PROTOZOA  1 29 

tiichinellac  when  their  capsules  are  digested  off  in  the  Kiistric  juice,  hut  comes  either 
from  the  free  larv;r,  or  from  the  dcRcncratcd  muscles  in  wliich  ihey  burrow. 
Coincident  bacterial  infection  may  reduce  the  number  of  eosinopliiles.  Ilerrick^ 
finds  that  extracts  of  Asciiris  Imnbricoidcs  cause  a  notable  eosinophilia,  but  only 
\vh(m  tlio  aninuil  has  been  sensitized  previously  with  the  same  extract,  tiie  active 
aKcnt  of  which  is  a  protein;  this  suggests  a  relationship  between  i)arasitic  and  ana- 
phylactic eosinoi)hilia.'  That  the  eosinophilos  i)lay  a  i)art  in  the  immunity 
reactions  observed  in  the  hosts  of  animal  parasites  is  indicaterl  by  the  fact  that 
hydatid  fluid  loses  its  antigenic  pr()])erti(>s  when  in  contact   with  eosinopliiles.* 

PROTOZOA 

These  uuicellular  I'orins  possess  all  the  chemical  characters  of  the 
cells  of  higher  forms,  even  to  the  more  specialized  constituents.  Thus 
it  has  been  demonstrated  that  protozoa  contain  proteolytic  enzymes,'" 
and  that  they  secrete  an  acid  into  their  digestive  vacuoles. '^  On  the 
other  hand,  Amoeba  coll  does  not  seem  to  digest  the  red  corpuscles  and 
the  bacteria  that  it  takes  up.'^  Whether  the  Amceha  coli  produces 
any  toxic  materials,  specific  or  non-specific,  has  not  yet  been  determined, 
but  the  necrosis  that  it  produces  in  liver  abscesses,  when  bactewal 
cooperation  can  often  be  excluded  by  culture,  strongly  indicates 
the  production  of  necrogenic  substances.  Apparently  these  sub- 
stances are  not  chemotactic,  in  view  of  the  absence  of  leucocytic  ac- 
cumulation in  the  lesions  of  amebic  dysentery'.  There  is  also  no 
evidence,  clinical  or  experimental,  that  amebic  infection  causes  the 
formation  of  anti-substances  of  any  kind  in  the  body  of  the  host. 
The  spontaneous  recovery  from  amebic  and  other  protozoan  infections, 
however,  may  be  considered  as  indicating  the  development  of  an 
immunity  against  these  organisms.'^  Numerous  observers  have  sug- 
gested the  possibility  of  obtaining  artificial  immunity  against  pro- 
tozoa, and  Rossle'^  has  obtained  immune  sera  against  infusoria. 

The  serum  of  rabbits  immunized  against  amoebae  was  foimd  b}' 
Sellards'^  to  be  cytolytic  for  the  same  amoebae,  but  no  antibodies 
could  be  foimd  in  the  blood  of  patients  with  amebic  dysentery.  The 
serum  of  persons  infected  with  bilharzia  is  said  to  give  specific  com- 
plement fixation  reactions  (Fairley) . ^^  Novy '^  has  obtained  immunity 
against  trypanosomes,  but  the  serum  of  immune  animals  will  not 
confer  passive  immunity.     Braun  and  Teichmann,'^  however,  claim 

^  Arch.  Int.  Med.,  1913  (11),  165. 

»  Supported  by  Paulian,  Presse  Med.,  1915  (23),  403. 

^  Weinberg  and  Seguin,  Ann.  Inst.  Pasteur,  1910  (30),  323. 

">  Mouton,  Compt.  Rend.  Soc.  Biol.,  1901  (53),  801. 

"  Le  Dantec,  Ann.  Inst.  Pasteur,  1890  (4),  776;  Greenwood  and  Saunders,  Jour, 
of  Phy,siol.,  1894  (16),  441. 

'-  Musgrave  and  Clegg,  Bureau  of  Gov't.  Laboratories,  Manila,  1904,  No.  18, 
p.  38. 

'^  Concerning  immunity  to  protozoanjinfections  see  Schilling,  KoUe  and  Wasser- 
mann's  Handbuch,  1913  (7),  566. 

'^  Arch.  f.  Hyg.,  1905  (54),  1;  full  review  of  this  topic. 

'5  Philippine  Jour.  Sci.,  1911  (6).  281. 

'» Jour.  Kov.  Armv  Med.  Corps,  1919  (32).  449. 

'"Jour.  Infec.  Dis.,  1912  (11),  411. 

'«  Zeit.  Immunitat.,  Ref.,  1912  (6),  465. 
9 


130  CHEMISTRY  OF  THE  ANIMAL  PARASITES 

positive  results  with  immune  serum  from  rabbits;  they  found  no  poison- 
ous agent  in  trypanosome  substance. ^^  The  fact  that  trypanosomes 
themselves  readily  become  immune  to  various  trypanocidal  chemicals 
has  been  demonstrated  and  extensively  studied  in  Ehrhch's laboratory. 
Gonder^o  j^^s  made  the  interesting  observation  that  trypanosomes 
which  can  be  stained  by  certain  vital  stains,  become  unstainable  while 
alive  if  immune  to  arsenic  compounds,  suggesting  that  this  immunity 
is  associated  with  considerable  structural  or  chemical  changes. 

Plasmodium  malariae  undoubtedly  produces  toxic  substances, 
which  seem  to  be  of  such  a  nature  that  they  do  not  diffuse  from  the  red 
corpuscle,  but  are  only  liberated  when  the  corpuscle  breaks  up  on 
the  maturation  of  the  parasite.  In  this  way  the  characteristic  par- 
oxj^smal  manifestations  of  the  disease  are  produced.  The  nature  of 
the  poison  or  poisons  is  unknown,  but  we  have  evidence  that  it  is 
hemolytic,  since  malarial  serum  may  hemolyze  normal  corpuscles, ^^ 
and  extracts  of  the  parasites  are  stronglj^  hemolytic  (Brem-^);  prob- 
ably the  malarial  hemoglobinuria  is  caused  by  this  hemolj-sis.  Pre- 
sumably malarial  poisons  are  not  extremely  toxic  for  parenchymatous 
cells,  since  the  parenchymatous  lesions  in  malaria  seem  to  be  relatively 
slight  as  compared  with  the  intensity  and  duration  of  the  intoxication. 
Some  authors  state  that  the  toxicity  of  the  urine  is  increased  after 
the  paroxysm,-^  which,  however,  does  not  necessarily  indicate  that 
a  poison  formed  by  the  parasites  is  excreted  in  the  urine.  Immunity 
seems  to  be  seldom  developed  against  the  malarial  poison  or  against 
the  parasite  itself,  although  some  persons  seem  to  be  naturally  im- 
mune, w^hile  some  acquire  immunity  through  previous  infection.-^ 
The  blood  of  persons  with  malaria  seems  to  contain  no  antibodies  for 
the  parasite  (Ferrannini),^^  although  it  seems  to  have  some  antihemo- 
lytic  power  (Brem).  (Concerning  the  pigment  present  in  the  ma- 
larial parasites  see  "Pigmentation,"  (Chap,  xviii). 

Sarcosporidia  of  sheep  yield  aqueous  and  glycerol  extracts  that 
are  highly  toxic  for  rabbits  (Pfeiffer),  the  poisonous  constituent  of 
which  was  called  sarcocystin  by  Laveran  and  Mesnil.-^  This  is  so 
highly  toxic  that  0.0001  gm.  is  fatal  to  rabbits  (per  kilo),  other  ani- 
mals being  less  susceptible.  It  loses  its  toxicitj''  on  heating  at  85° 
for  twenty  minutes,  and  is  impaired  at  55-57°  for  two  hours.     It 

1"  Hintze  (Zeit.  f.  Hyp;.,  1915  (80),  377)  obtained  little  immunity  with  T. 
brucei,  hut  Scliiiling  and  Kondoni  (Zeit.  Imiminitat.,  1913  (IS).  t)51)  obtained'a 
poison  from  Nagana  trypanosomes  which  produced  active  imnumity  in  mice. 
When  trypanosomes  are  killed  by  weak  electric  currents  they  may  liberate  an 
active  poison  (Uhlenhutli  and  Seyderhelni,  Zeit.  Immimitjit.,  1914  (21),  366). 

"Zeit.  Immunitilt.,  1913  (15),  257. 

"  See  Regnault,  Kevue  de  MM.,  1903  (23),  729. 

"Arch.  Int.  Med.,  1912  (9).  129. 

"(Quoted  from  HIanchurd,  Arch.  d.  Parasitol.,  1905  (10),  83;  this  article  gives 
a  r6sum('  of  tlie  siibj(!c(  of  the  iox'w.  substances  produced  by  the  animal  parasites. 

"  See  Celli,  Cent.  f.  Hakt.,  1900  (27),  107. 

"  Kiforma  Med.,  1911  (27),  177. 

=»  Compt.  Rend.  Soc.  Bio!.,  1899  (51),  311. 


CESTODES  1  '4 1 

produces  pruritis  ami  otluu-  aiiapliylactic  symptoms,  and  altliouf^h  the 
serum  of  sheep  with  this  parasite  does  not  confer  passive  anaphylaxis 
to  sarcosporidia,  yet  it  does  give  positive  complement  fixation."  That 
it  is  a  true  toxin  is  shown  by  Tcichmann  and  Braun,-**  who  produced 
an  effective  antitoxin  by  immunizing  rabbits;  only  rabbits  seem  to 
be  susceptible  to  the  toxin.  The  sarcosporidia  contain  also  a  distinct 
thermostable  agglutinin.  The  lethal  dose  of  dried  substance  of  sar- 
cosporidia is,  for  rabbits,  but  0.0002  gm.,  and  the  poison  seems  to  unite 
with  the  lipoids  of  the  nervous  system  (Teichmann).^^  It  is  probable 
that  the  pathogenic  protozoa,  at  least  in  some  instances,  have  a  semi- 
permeable membrane  about  them,  for  GoebeP''  found  that  trypano- 
somes  are  very  susceptible  to  changes  in  osmotic  conditions. 

rCESTODES] 

Taenia  echinococcus  has  been  by  far  the  most  studied,  its  abundant 
fluid  content  furnishing  suitable  material  for  investigation.  That 
this  fluid  is  toxic  has  been  repeatedly  observed  when,  through  rup- 
ture or  puncture,  the  fluid  has  escaped  into  the  body  cavities;  such 
accidents  are  often  followed  by  violent  intoxication,  sometimes  by 
death. 3^  As  long  as  the  cyst  is  unopened  no  toxic  manifestations  are 
observed.  The  most  constant  symptoms  are  local  irritation  and  in- 
flammation, accompanied  by  urticaria,  which  may  also  be  produced 
experimentally  in  man  if  the  cyst  contents  are  injected  subcutaneously. 

The  symptoms  are  so  strikingly  similar  to  those  of  anaphylactic 
intoxication,  that  it  is  now  generally  believed  that  they  are  the  result 
of  such  a  reaction  in  a  person  sensitized  by  absorption  of  antigenic 
substances  from  the  cyst.^^  Carriers  of  echinococcus  cysts  have  been 
found  to  have  in  their  blood  antibodies  giving  precipitins^  and 
complement  fixations'*  reactions  with  extracts  of  echinococcus,  and 
sometimes  with  other  taenia. ^^  The  antigen  of  the  echinococcus  is  be- 
lieved by  some  to  be  a  lipoid ;S6  in  the  case  of  Taenia  saginata,  at  least, 
it  seems  to  be  associated  with  the  lecithin  (JMeyer^^).  Graetz," 
however,  states  that  the  protein  of  the  hydatid  cyst  is  derived  from 
the  host,  and  that  it  is  therefore  incapable  of  causing  anaphylaxis  in 
that  host,  but  it  may  undergo  alterations  in  the  cyst  so  that  it  is 
toxic  after  the  order  of  anaphylatoxins  (q.  v.) .     The  complement  fixa- 

"  McGowan,  Jour.  Path,  and  Bact.,  1913  (18),  125. 

28  Arch.  f.  Protistenk.,  1911  (22),  351. 

"  Ibid.,  1910  (20),  96;  see  also  Knebel,  Cent.  f.  Bakt.,  1912  (66),  523. 

30  Ann.  Soc.  Mrd.  d.  le  Gand,  1906  (86),  11. 

3'  See  Achard,  Arch.  gen.  de  Med.,  1887  (22),  410  and  572. 

^^  See  Boidin  and  Laroche,  Presse  Med.,  1910  (18),  329;  Ghedini  and  Zamorani, 
Cent.  f.  Bakt.,  1910  (55),  49. 

"  Welch,  et  a/.,  Lancet,  1909,  Apr.  17. 

"  Kreuter,  Miinch.  med.  Woch.,  1909  (56),  1828;  Weinberg,  Ann.  Inst.  Pasteur 
1909  (23),  472. 

3^  Meyer,  Berl.  klin.  Woch.,  1910  (47),  1316;  Zeit.  Immiinit.nt.,  1910  (7),  732. 

3«  Israel,  Zeit.  Hyg.,  1910  (66),  487;  Meyer,  Zeit.  Immunitat.,  1911  (9),  530. 

3'  Zeit.  Immunitat.,  1912  (15),  60;  general  review. 


132  CHEMISTRY  OF  THE  AMMAL  PARASITES 

tion  reaction  with  echinococcus  fluid  has  been  found  quite  rehable  in 
the  cHnic,  93  per  cent,  of  positive  reactions  having  been  obtained  in 
500  cases  collected  by  Zapelloni,'^  while  controls  were  always  negative. 

The  fluid  of  the  echinococcus  cysts  has  generally  a  specific  gravity 
of  1005-1015,  and  contains  1.4-2  per  cent,  of  solids.  Most  abundant 
are  sodium  chloride,  about  0.8  per  cent.,  and  sugar,  0.25  per  cent., 
the  latter  presumably  coming  from  the  glycogen  contained  in  the 
wall.  Cholesterol  is  often  abundant,  while  inosite,  creatin,  and  suc- 
cinic acid  are  often  found.  Clerc  has  found  traces  of  lipase,  but 
other  enzj'^mes  seem  to  be  absent  or  in  very  small  amounts.  Proteins 
are  present  only  in  traces,  unless  inflammation  has  occurred.  Schil- 
ling''^ found  the  molecular  concentration  of  the  cyst  fluid  to  be  quite 
the  same  as  that  of  the  patient's  blood.  The  fluid  is  said  not  to  be 
toxic  to  laboratory  animals.*" 

The  cyst  wall  consists  of  a  hyaline  substance  which  seems  to  stand 
between  chitin  and  protein,  and  probably  consists  of  a  mixture  of 
both.  Because  of  the  chitin  it  yields  about  50  per  cent,  of  a  reducing, 
sugar-like  body  when  boiled  with  acid.  Glj'cogen  is  also  usually 
present,  but  it  is  limited  to  the  germinating  membrane."'^ 

Other  cestodes,  when  in  the  cystic  form,  contain  fluids  which  are 
more  or  less  toxic.  Thus  Moursou  and  SchlagdenhaufTen*-  found  a 
"leucoma'in"  in  the  Cy.sticercus  tenuicoUis,  the  larva  of  Taenia  'mar- 
ginata,  which  causes  urticaria  and  other  toxic  sjaiiptoms  when  in- 
jected into  animals  (thus  resembling  histamine).  The  fluids  of 
Cysticercus  pisifonnis  (the  common  cestode  of  rabbits)  have  been 
found  toxic  for  frogs,  and  Vaullegeard*^  has  determined  the  presence 
of  an  "alkaloid"  and  a  "ferment  toxin"  in  this  fluid.  The  fluids  of 
the  cysts  of  Ccenurus  cerebralis,  Ccenurus  serialis,  and  Echinococcus 
polymorphus  have  all  been  found  toxic,  and  it  is  probable  that  this  is  a 
general  rule  with  the  cestodes,''''  but  human  forms  other  than  the  echi- 
nococcus seem  not  to  have  been  investigated;''''  according  to  Jnmmes 
and  Mandoul,  extracts  of  taenia  are  bactericidal.^" 

Dibothriocephalus  latus  ficqucntly  causes  anemia,  which  has  been 
attributed  to  a  poison  liJK'rated  by  the  parasite  when  it  undergoes 
disintegration,  and  possibly  as  a  secretion  of  the  living  worm.'*^  All 
the  intestinal  cestodes  iire  equipped  with  a  well-developed  excretory 
apparatus,  and  it  is  easy  to  imagine  that  their  excretory  prochicts 
may  be  toxic  to  tlic  animal  into  whose  intestine  they  ai'e  exereteil. 

3»  Policlinicu,  Suim;.,  liKf)  (22j,  \us.  ti    11. 

3»Cent.  inn.  iMid.,  1904  (2.'3),  HXi. 

■"•Uraetz.  Cent.  f.  IJukt.,  1910  (.Wj,  2:U;  Zi-it.  Iniiiuinitat.  1912  (15),  60. 

^'  Hrault  und  Loupcr,  .K)ur.  I'livs.  et.  Tatli.  m''n.,  H'Ol  (ti),  295. 

^■-Coinpt.  Hcnd.  S(.c.  Bio!,,  I8S2  (95),  791. 

■••'  Bull.  S(K'.  liniu'cniic  (li?  Nurinaiulie,  1901  (l),  SI. 

"  HhuK-liaid.,  Iin-  ril.-' 

■"•  Scniaiai-  iiu'd.,  1905  (25j,  .55. 

^«  See  alo  .Joycu.x,  .\rcli.  d.  I'aiasitol.,   1907  (ID,  409. 

*''  Litt'ia  lire  l)\-   Kl;iiu-liai(l,  lor  <•//.-■' 


CESTODES  133 

Talhivist^"'  has  made  oxIcMisivc  studies  of  Ix)!  liiioccplialns,  whicli  show 
that  the  active  hemolytic  agent  is  contaiiuMl  In  the  lii)oids  of  the 
parasites,  presumably  as  a  cholesterol  ester  of  oleic  acid."*"  The 
j)roglottidcs  contain  a  proteolytic  enzyme,  which  apparently  digests 
the  substance  of  dead  segments,  liberating  the  hemolytic  lipoid,  which 
constitutes  about  ten  per  cent,  of  the  solids  of  the  parasite.  There 
is  also  a  hemagglutinin,  which,  unlike  the  hemolytic  substance,  is  ther- 
molabile,  and  causes  the  appearance  of  an  antibody  in  immunized 
animals.  In  common  with  other  parasites,  antitryptic  and  antijieptic 
effects  are  exhibited  by  extracts. 

Rosenqvist'"  has  studied  the  metabolism  of  twenty-one  cases  of 
bothriocephalus  anemia,  and  found  evidence  in  nearly  all  of  a  toxo- 
genic  destruction  of  protein,  which  ceases  promptly  when  the  worms 
are  removed.  He  has  found  that  these  worms  produce  a  poison  which 
is  globulicidal,  and  probably  also  generally  cytotoxic,  since  in  the 
anemias  that  they  produce,  the  elimination  of  purine  bodies  of  tissue 
origin  (endogenous  purine)  is  increased.  The  nitrogenous  metabolism 
is  quite  the  same  in  pernicious  anemia  and  in  ])othriocephalus  anemia. 
Isaac  and  v.  d.  Velden"'  state  that  the  blood  of  patients  infected  with 
this  parasite  gives  a  predpitin  reaction  with  autolytic  fluid  obtained 
from  bothriocephalus,  and  that  rabbits  immunized  with  such  autolytic 
fluids  developed  a  precipitin.  Complement  fixation  relictions  may  be 
demonstrated  in  human  infections  with  bothriocephalus  or  other 
taenia  (Jerlov).^^" 

Other  Taenia. — There  is  much  less  evidence  that  other  forms  of  tffuia  produce 
toxic  substances  which  injure  their  host,  although  the  clinical  manifestations  ob- 
served in  persons  harboring  tajnia  are  often  of  such  a  nature  as  to  indicate  strongly 
an  intoxication.  Jammes  and  Mandoul*-  found  no  toxic  manifestations  produced 
by  extracts  of  Tcenia  saginata,  which  negative  finding  is  supported  by  Cao,^'  Tall- 
qvist  and  Boycott,"  using  various  sorts  of  taenia.  These  results  contradict  the 
earlier  positive  findings  of  Messineo  and  Calamida,^^  who  found  extracts  of  taenia 
from  dogs  to  be  hemolytic,  chemotactic  (especiallj^  for  eosinophiles),  and  to  cause 
local  fatty  degeneration  in  the  liver.  Extracts  of  T.  perjoliata  and  plicata  (of  the 
horse)  were  found  highly  toxic  for  guinea-pigs  by  Pomella,^'  the  liematopoietic 
organs  being  greatly  stimulated.  Bedson"  found  that  extracts  of  all  sorts  of 
helminths  produced  similar  effects  on  guinea-pigs,  the  chief  lesions  being  in  the 
adrenals  and  thyroid.  Possibly  these  differences  in  results  are  due  to  the  fact 
that  different  parasites  were  studied  by  different  investigators;  furthermore,  tests 
of  toxicity  of  human  parasites  upon  rabbits  and  guinea-pigs  can  haidly  be  con- 
sidered conclusive.  Le  Dantec  did  not  find  a  precipitin  for  T(snia  saginata  ex- 
tracts in  the  blood  of  persons  harboring  this  parasite,  and  negative  results  with 

«  Zeit.  klin.  Med.,  1907  (61),  427. 

*^  Faust  and  Tallqvist,  Arch.  exp.  Path.  u.  Pliarm.,  1907  (57),  307. 

^o  Zeit.  klin.  Med.,  1903  (49),  193. 

5>  Dent.  med.  Woch.,  1904  (30),  982. 

s'"  Zeit.  Immunitat.,  1919  (28),  489. 

"Conipt.  Rend.  Acad.  Sci.,  1904  (138),  1734. 

"  Riforma  med.,  1901  (3),  795. 

^  Jour.  Pathol,  and  Bacteriol.,  1905  (10),  383. 

«  Cent.  f.  Bakt.,  1901  (30),  346  and  374. 

«  Compt.  Rend.  Soc.  Biol.,  1912  (73),  445. 

"  Ann.  Inst.  Pasteur,  1913  (27),  682. 


134  CHEMISTRY  OF  THE  ANIMAL  PARASITES 

several  other  taenia  were  obtained  by  Langer,^*  but  complement  fixation  reactions 
may  be  given/* 

Picou  and  Ramond^"  state  that  tirnia  extracts  undergo  putrefaction  very 
slowly,  and  attribute  this  to  a  bactericidal  property,  which  was  observed  with 
several  forms  of  ttmia  by  AUesandrini.  Weinland^'  has  found  that  many  intes- 
tinal parasites  exhibit  antitnjplic  properties, ^^  but  in  a  study  of  the  histological 
changes  of  autolysis  I  observed  a  t  enia  in  the  intestine  of  a  dog  undergo  more 
rapid  karyolytic  changes  than  did  the  intestinal  epithelium.  Dastre  and  Stessano*' 
state  that  extracts  of  Taenia  serrata  act  upon  enterokinase  rather  than  on  tryp- 
sinogen. 

NEMATODES 

Ascaris. — The  toxicity  of  members  of  this  group  has  been  a  matter 
of  dispute,  although,  as  with  the  Taenia,  there  have  been  observed  in 
patients  symptoms  that  ^vere  more  easily  explained  as  due  to  chemical 
substances  than  as  due  to  mechanical  irritation.  Miram,  while 
studying  Ascaris  megaloceyhala,  suffered  from  attacks  of  sneezing, 
lachrymation,  itching,  and  swelling  of  the  fingers,  v.  Linstow  suffered 
from  a  severe  attack  of  conjunctivitis  with  chemosis  after  touching  his 
eye  with  a  finger  that  had  been  in  contact  with  one  of  these  worms. 
Others  have  had  similar  experiences,  and  it  has  been  found  that  the 
fluid  from  these  worms  is  toxic  to  rabbits.  In  man  it  seems  to  affect 
especially  those  who  have  been  sensitized  by  previous  poisoning,  some 
persons  being  entirely  insusceptible. 

An  extensive  investigation  of  ascaris  from  both  the  chemical  and 
toxicological  standpoint  has  been  made  by  Flury,^*  which  indicates 
the  source  and  nature  of  these  toxic  substances.  Because  of  the 
practically  anaerobic  conditions  under  which  the  worms  live,  Flury 
believes,  the  products  of  their  metabolism  are  charactetized  by  being 
incompletely  oxidized,  and  resemble  the  products  of  anaerobic  bac- 
teria. Most  important  of  these  are  volatile  aldehj'des  and  fatty  acids, 
especially  valerianic  and  butyric  acids,  in  less  quantities  formic, 
acrylic  and  propionic  acids.  The  toxicologic  action  of  these  volatile 
substances  is  of  such  a  character  as  fully  to  explain  the  severe  irrita- 
tion of  skin  and  mucous  membranes  observed  in  persons  handling 
these  parasites;  aldehydes  are  notoriously  inclined  to  produce  con- 
ditions of  hypersensitivenoss,  c.  r/.,  formaldehyde.  It  is  quite  jiossi- 
ble  that  the  severe  constitutional  symptoms  observed  occasionally  in 
persons  infected  with  ascaris,  are  produced  by  these  substances  or 
by  poisonous  substances  set  free  through  disintegration  of  worms 
which  have  dietl  and  renuiined  in  the  ])()wcl.  A  capillarv  poison  re- 
sembling scpsin,  poisonous  bases   acting   lik(>  atropine  and  coniine, 

"  Munch,  mod.  Woch.,  1905  (.W),  1(H)5. 
'»  Mover,  Zoit.  Immunit;it.,  1<H0  (7),  732. 
"o  Comi)t.  Rend.  Soc.  iiiol.,  1S<M)  (f)!),  17G. 
9|  Zoit.  f.  Hiol.,  11)02  Ml),  1  and  -ir). 

'-  Corrcjboratod  for  'I'druii  /iiuiinnta  by  Fetterolf  (I'niv.  of  IVnnsvlvania  Mod. 
Bull.,  1907  (20),  91). 

«^  Compt.  liond.  Soc.  Hiol.,  1903  (55),  130. 

"'Arch.  oxp.  I'alh.  ii.  I'li.i,  iii.,  1<)12  (07),  275  (litonituro). 


NEMATODES  135 

and  lieniolytic  unsaturated  fatty  acids  were  also  found,  among  other 
less  toxic  substances  produced  by  ascaris,  and  tin;  sum  of  their  action 
is  certainly  adequate  to  account  for  anything  ascribed  to  these  para- 
sites. Paulian,"  however,  would  attribute  the  chief  effect  to  ana- 
phylaxis from  absorbed  proteins,  while  Brinda^®  believes  that  ascaris 
produces  an  active  toxalbumin.  This,  he  found,  causes  a  tetany- 
hkc  tj'^pe  of  respiration,  and  a  similar  symptom  is  often  noticed  in 
chiklren  with  ascarides.  An  actively  toxic  mixture  of  proteoses  and 
peptones  has  been  obtained  from  several  species  of  ascaris,  and  desig- 
nated as  "askaron,"  by  Shimamura  and  Fujii.^^  Horses  can  be 
immunized  to  withstand  400  lethal  doses.  Ether  and  alcohol  ex- 
tracts of  ascaris  are  not  poisonous  in  large  doses,  although  they  are 
hemolj''tic. 

Analysis  of  a  great  quantity  of  ascaris  from  horse  and  hog  gave  as  the  chief 
"•esiilts,  the  following :'^^  They  differ  much  in  composition  from  the  higher  ani- 
mals. About  half  the  ash  is  water  soluble;  and  of  the  dry  substance  about  half 
is  protein  or  related  substances,  from  which  the  usual  amino-acids  and  purines 
can  be  isolated.  Uric  acid  and  creatinin  were  lacking.  The  superficial  layer  does 
not  consist  of  chitin,  but  of  an  albuminoid  rich  in  sulphur  and  free  from  carbohy- 
drates, resembling  keratin.  They  have  abundant  and  active  enzymes  of  many 
kinds.  Glycogen  is  the  chief  carbohydrate,  but  there  are  also  glucoproteins  and 
glucose.  The  ascaris  differs  from  higher  animals  especially  in  the  ether-soluble 
substances,  which  consist  chiefly  of  free  fatty  acids,  many  of  which  are  volatile. 
Also  found  were  lecithin,  aldehydes  and  neutral  fats,  but  little  glycerol,  no  chol- 
esterol, and  an  "ascaryl  alcohol"  (C32H64O2)  which  probably  substitutes  for  both 
glycerol  and  cholesterol. 

Trichinella  Spiralis  has  been  investigated  from  the  chemical  stand- 
point by  Flury,^^  who  found  that  the  infected  muscles  of  experimental 
animals  differed  from  normal  muscles  in  having  more  water  because  of 
edema,  an  increase  in  extractives,  ammonia  compounds,  lactic  acid  and 
volatile  acids,  with  fluctuating  values  in  both  creatine  and  purines. 
Glycogen  is  decreased  not  only  in  the  infected  muscle  but  also  in  the 
liver  and  kidneys.  The  parasites  themselves  are  remarkably  resist- 
ant to  strong  acids,  perhaps  because  of  the  lipoid  content  of  their 
surface  covering,  in  which  keratin  could  not  be  positively  identified; 
cholesterol  and  glycogen  were  present.  The  blood  of  infected  ani- 
mals shows  an  excess  of  nuclein  material,  and  may  give  albumose  and 
diazo  reactions;  the  red  corpuscles  have  a  lowered  resistance  to  hemo- 
lysis by  hypotonic  solutions.  Trichinous  muscle  contains  substances 
that  produce  marked  local  tissue  irritation,  which  may  be  purines;  a 
curare-like  poison  was  also  found,  which  was  believed  to  be  a  guanidine 
derivative,  as  well  as  a  "fatigue  poison"  which  probably  consists  of  the 
lactic  acid  and  other  muscle  extractives.  The  location  of  trichinella 
in  muscle  may  be  ascribable  to  their  need  for  glycogen  for  nourish- 
es Compt.  Rend.  Soc.  Biol.,  1915  (78),  73. 
««  Arch,  de  Med.,  1915  (17),  SOI. 

"  Japanese  Jour.  Bact.  (Saikingaku  Zassi),  June  10,  1916. 
"  Arch.  exp.  Path.  u.  Pharm.,  1913  (73),  164  and  214. 


13G  CHEMISTRY  OF  THE  ANIMAL  PARASITES 

ment  and  the  fact  that  their  metabolism  is  carried  out  anaerobically 
may  account  for  the  character  of  the  products  (fatt}-  acids,  etc.)- 

The  intoxication  of  trichinosis  probably  is  the  combined  result  of 
the  products  of  the  metabolism  of  the  parasites,  the  products  of  muscle 
disintegration,  and  perhaps  also  of  anaphylactic  reaction  to  the  pro- 
teins of  the  parasite  and  the  altered  muscle  proteins.  As  evidence  of 
the  anaphylactic  condition  is  the  conspicuous  eosinophilia,  which  we 
know  is  often  the  result  of  anaphylactic  intoxication.^^  Metabolism 
studies  show  a  preliminar}^  nitrogen,  creatinine  and  purine  retention, 
followed  by  excessive  loss  of  all  three.  There  is  also  an  intense  diazo 
reaction,  and  increased  excretion  of  lactic  and  organic  acids.  The 
hypothesis  that  bacterial  invasion  is  responsible  for  the  intoxication 
of  trichinosis  does  not  seem  to  be  well  supported  (Herrick,  Gruber). 

The  serum  of  infected  animals  is  not  toxic,  and  does  not  protect 
against  infection  with  trichinella  (Gruber'").  Salzer,^^  however, 
found  that  the  serum  of  recovered  patients  had  a  curative  effect  in 
persons  acutely  intoxicated  with  trichiniasis,  and  also  a  marked  pro- 
phylactic effect  in  experimental  animals ;^2  it  removed  the  eosinophilia 
both  in  men  and  animals.  He  also  observed  evidence  of  a  reduction 
of  the  bilirubin  of  the  feces  by  the  trichinae,  so  that  the  stools  were 
clay  colored  without  icterus.  Positive  complement  fixation  reactions 
are  given  by  the  serum  of  trichinella  infected  persons." 

Uncinaria  duodenalis,  which  has  for  its  chief  effect  the  production 
of  a  severe  anemia,  seems  to  cause  this  anemia  by  producing  repeated 
small  hemorrhages  rather  than  by  any  toxic  action.  The  abundance 
of  this  loss  of  blood  is  explained  by  L.  Loeb^'*  as  due  to  the  presence, 
in  the  anterior  portion  of  the  parasite  (they  studied  Ankijlostoma 
caninum.),  of  a  substance  that  inhibits  the  coagulation  of  the  blood. 

However,  Preti^^  would  ascribe  importance  to  a  lipoidal  or  lijund- 
like  hemolytic  constituent  of  the  parasitic  tissues  of  the  European 
ankylostoma,  but  Whipple, ''"  who  has  observed  a  weak  hemolysin  in 
the  American  hook  worm,  considers  it  too  ineffective  to  be  of  practical 
importance.  In  Sclerosto7na  equinwn,  however.  Bondonoy''^  found 
active  hemolj^tic  agents,  ascribed  by  him  to  lipase;  also  a  ptomain,  an 
alkaloid  and  other  substances.  Corresponding  to  Flury's  analyses 
of  ascaris,  he  found  that  the  cuticle  is  albuminoid  and  not  chitinous, 

"'  See  Hcrrick,  Jour.  Amer.  Metl.  Assoc,  1915  (65).  1870;  Schwartz,  Ergeb. 
allg.  Pathol.,  1914  (17),  136. 

'»  Miinch.  mod.  Woch.,  1914  (61),  645. 

"  Jour.  Amer.  Med.  As-.soi\,  1916  (67),  579. 

"  Not  oorrohonilcd  by  Schwartz,  Jour.  Auum'.  Mvd.  Assoc,  1917  (69),  884; 
or  Hall  and  Wi^dor,  Arcli.  Int.  Med.,  191S  (22),  601. 

"Stroebel,  Munch,  ined.  Woch.,  1911  (.")S),  672. 

'UVnt.  f.  Hukt.,  1904  (37),  93;  1906  (40),  740;  IahA>  ami  FloLscher,  Jour. 
Infec.  i)i.s.,  1910  (7),  ()25. 

"  Miinch.  incd.  Woch.,  1908  (55),  436. 

'"Jour.  K\\).  Med.,  1909  (11),  331. 

"  .Vrch.  Parasitol.,  1910  (14),  5;  see  also  Ashcrolt,  C'onipt.  Jientl.  Soc.  Biol., 
1914  (77),  442. 


NEMATODES  137 

and  that  the  parasite  prochices  inucli  vohitilo  fatty  acids,  especially 
butyric;  both  lecithin  and  cholesterol  were  absent.  The  dermatitis 
produced  by  uncinaria  larvie  is  ascribed  by  C.  A.  Smith^**  to  an  alco- 
hol-soluble substance.  Watery  extracts  of  Sclerostoma  were  found  by 
Grosso^*  to  cause  but  slight  chemotaxis  without  eosinophilia. 

Filaria  seem  not  to  produce  any  apj)reciabl(!  amount  of  toxic  ma- 
terial, if  we  may  judge  by  the  slight  evidence  of  intoxication  shown 
by  infected  incUviduals.  Jin  exception  may  be  made  in  the  case  of 
the  guinea-worm  (Dracunculus  or  F.  medinensis).  This  parasite 
causes  chiefly  mechanical  injury  unless  its  bodj^  is  ruptured,  which 
may  happen  in  attempting  to  remove  it  forcibly;  this  accident  is  fol- 
lowed by  violent  local  inflammation  or  gangrene,  which  indicates 
that  some  powerfully  irritant  substance  is  liberated  from  the  torn 
body  of  the  worm.**" 

"8  .lour.  Amer.  Med.  Assoc,  1906  (47),  1693. 

"  Folia  Hematol.,  1912  (14),  18. 

8"  Earthworms  are  said  by  Yagi  (Arch,  internat.  pharmacodyn.,  1911  (21), 
105)  to  contain  a  hemoljrtic  substance,  "lumbricin,"  the  properties  of  which  he 
describes.  Nukada  and  Tenaka  (Mitt.  med.  FakuU.,  Tokio,  1915  (14),  1),  found 
an  antipyretic  agent  which  seems  to  be  derived  from  tyrosine. 


CHAPTER  VI 

PHYTOTOXINS  AND  ZOOTOXINS 

The  production  of  substances  possessing  the  essential  features  of 
true  toxins  is  by  no  means  limited  to  the  bacterial  cell.  In  the  plant 
kingdom  such  substances  are  formed,  and  called  phytotoxins.  Of 
these,  the  best  known  are  ricin,  abrin,  crotin,  and  robin.'  Among 
the  toxins  of  animal  origin,  zootoxins,  are  the  venoms  of  poisonous 
snakes,  lizards,  spiders  and  scorpions,  and  the  serum  of  eels  and  snakes. 

PHYTOTOXINS  2 

The  chief  phytotoxins  are  as  follows: 

Ricin,  from  the  castor-oil  bean  {Ricinus  communis). 

Abrin,  from  the  seeds  of  Ahrus  precatorius. 

Crotin,  from  the  seeds  of  Croton  tiglium. 

Robin,  from  the  leaves  and  bark  of  the  locust,  Rohinia  pseudoacacia. 

Curcin,  from  the  seeds  of  Jatropha  curcus. 

In  their  general  properties  all  these  substances  are  very  similar  and 
may  be  considered  together.  They  resemble  proteins  in  many  re- 
spects, for  they  can  be  salted  out  of  solutions  in  definite  fractions 
of  the  precipitate,  are  precipitated  by  alcohol,  and  are  slowly  de- 
stroyed by  proteolytic  enzymes.  For  some  time  they  were  referred 
to  in  the  literature  as  toxalbumins,  until  Jacoby  stated  that,  by  com- 
bining the  salting-out  method  with  trypsin  digestion,  he  was  able 
to  secure  preparations  of  ricin  and  abrin  that  did  not  give  the  pro- 
tein reactions.  This  seemed  to  place  them  in  the  same  category 
with  bacterial  toxins  and  enzymes,  i.  e.,  large  molecular  colloids,  closely 
resembling  the  proteins  with  which  they  are  associated,  but  still  not 
giving  the  usual  protein  reactions.  Because  of  their  great  similarity 
to  bacterial  toxins  this  seemed  a  very  probable  description,  and  it  has 
been  generally  accepted.  More  recent  work  by  Osborne,  Mendel,  and 
Harris,-*  however,  does  not  support  Jacoby's  contention.  They  found 
the  toxic  properties  of  ricin  associated  inseparably  with  the  coagulable 
albumin  of  the  castor  beans,  and  were  able  to  isolate  it  in  such  purity 
that  one  one-thousandth  of  a  milligram  (0.000001  gram)  was  fatal  per 
kilo  of  rabbit,  and  solutions  of  0.001  per  cent,  would  agglutinate  red 

1  The  poison  in  certain  i>oas,  especially  Lnthijrns  saluris,  which  causes  severe 
periphenil  i)aialvsis,  ('(ilhi/ri.sm)  is  believed  to  he  an  alkaloid.  (liXill  discussion  by 
Stockman,  I'^diiih.  Med.  Jour.,  1917  (10),  277). 

2  Ji.'suini'  of  literature  by  Ford,  Cent.  f.  Hakt.,  1913  (58),  129;  Jacoby,  Kolle 
and  Wass(M-inann's  IIundbu(;Ii,  1913  (2),  M.')3. 

•'  Atiier.  Jour,  of  IMiysiol.,   190.")  (14),  259. 

13S 


IMMUNITY  AGAINST  PIIYTOTOXINS  139 

corpuscles.  The  toxicity  was  also  impaired  or  destroyed  by  tryptic 
digestion.  They  consider  that  probably,  because  of  its  extremely 
great  toxicity,  Jacoby  was  able  to  get  active  preparations  that  con- 
tained too  little  active  substance  to  give  the  protein  reactions.  As 
they  remark:  "If  one-thousandth  of  a  milligram  of  a  compound 
giving  on  analysis  every  indication  of  being  a  relatively  pure  protein, 
is  physiologically  active  in  the  degree  characterized  by  our  experi- 
ments, the  toxicity  of  any  impurity  must  be  infinitely  greater  than 
that  of  any  known  toxins."  Against  the  claim  that  the  toxic  principle 
is  simply  carried  down  with  the  protein  is  the  fact  that  it  does  not  come 
down  in  the  first  fraction  that  is  precipitated,  the  globulin,  which  usu- 
ally carries  down  all  ipipurities.  All  the  ricin  comes  down  betv\een 
the  limits  of  one-fifth  and  one-third  saturation  with  ammonium  sul- 
phate, exactly  as  does  the  albumin.  During  germination  of  the  castor 
bean  the  ricin  disappears  with  the  albumin.^  Field^  has  found  evi- 
dence that  the  agglutinin  and  toxin  of  pure  ricin  are  separable,  but 
Reid  believes  them  identical.  Of  21  varieties  of  ricinus  seeds  ex- 
amined by  Agulhon,^  all  yielded  hemagglutinins.  Ricin  agglutinates 
not  only  corpuscles,  but  tissue  cells  of  all  sorts,  and  causes  precipitates 
in  normal  serum. '^  Curcin  alone  seems  to  have  no  hemagglutinative 
action.'* 

Immunity. — The  phytotoxins  have  been  very  serviceable  in  the 
study  of  immunity,  since  thej^  obey  the  same  laws  as  bacterial  toxins 
and  can  be  handled  in  more  definite  quantities.  By  their  use  Ehrlich 
first  determined  that  toxin  and  antitoxin  act  quantitatively.  They 
seem  to  possess  haptophore  and  toxophore  groups,  and  immunity  is 
readily  obtained  against  them,  not  only  by  subcutaneous  injection, 
but  by  dropping  into  the  conjunctival  sac,  and  also  by  feeding,  show- 
ing their  direct  absorbability  and  their  resistance  to  digestion.  The 
antitoxin  is  present  in  the  milk  of  the  immunized  mother  and  im- 
munizes the  suckling;  but  little  is  carried  through  the  placenta  into 
the  fetal  blood.  The  immunity  is  specific,  ricin  antitoxin,  for  exam- 
ple, not  protecting  against  abrin  (although  it  is  said  to  protect  against 
robin).  Roemer  found  that  in  animals  immunized  by  conjunctival 
application  the  eye  so  used  became  immune  to  the  local  action  of  the 
poison  before  the  other  eye  did,  indicating  a  local  development  of 
immune  substance.  In  general  immunization  the  immune  substance 
appears  first  in  the  spleen  and  bone-marrow.  Normal  serum  gives 
a  precipitate  with  ricin,  but  immune  serum  gives  a  much  heavier  one. 
Antiricin,  like  other  antitoxins,  is  inseparable  from  the  proteins  of  the 
serum. 

^  Agulhon,  Ann.  Inst.  Paste-ir,  1915  (29),  237. 

5  Jour.  Exper.  Med.,  1910  (12),  551;  Reid,  Landwirtsch.  Versuchst.,  1913  (82), 
393. 

«  Ann.  Inst.  Pasteur,  1914  (28),  819. 

'  Michaelis  and  Steindorff,  Biochem.  Zeit.,  1906  (2),  43. 

«  Felke,  Landwirts.  Versuchst.,  1913  (82),  427. 


140  PHYTOTOXINS  AXD  ZOOTOXINS 

Physiological  Action. — Their  poisonous  action  is  manifold,  most 
prominent  being  agglutination  of  the  erj'throcytes,  local  cellular  de- 
struction, and,  to  a  less  extent,  hemolysis.  Jacoby  believes  that  in 
ricin  there  are  several  toxic  substances  differing  in  physiological  prop- 
erties, similar  to  Ehrlich's  findings  in  diphtheria  toxin  (toxones, 
etc.).  B}'-  long  action  of  pcpsin-HCl  upon  ricin,  he  secured  a  prepa- 
ration with  all  the  other  properties  of  ricin  except  that  it  was  inactive 
against  erythrocytes;  the  same  result  could  not  be  obtained  with 
abrin.  Heating  to  65°  or  70°  does  not  destroy  the  toxicity  of  phy- 
totoxins,  but  boiling  does.  There  is  a  latent  period  of  several  hours 
after  injection  of  the  poison,  the  onset  of  sj'mptoms  being  sudden; 
death  rarely  occurs  in  less  than  fifteen  to  eighteen  hours  (Osborne 
et  al.). 

Flexner^  has  studied  particularly  the  histological  changes  pro- 
duced by  ricin  and  abrin  poisoning  in  animals.  Both  act  alike,  af- 
fecting the  tissues  much  as  bacterial  toxins  do  (diphtheria).  Fever, 
albuminuria  and  convulsions  are  followed  by  exhaustion  and  lowered 
temperature.  Punctiform  hemorrhages  are  found  beneath  the  serous 
surfaces,  with  fluid  in  the  peritoneal  cavity.  At  least  in  the  case 
of  ricin  the  hemorrhages  are  not  due  to  blood  changes,  but  to  a  spe- 
cial toxin  destroying  the  endothelial  cells. ^^  There  occur  a  general 
lymphatic  enlargement  and  marked  changes  in  the  intestinal  mucosa, 
with  swelling  of  the  Peyer's  patches.  The  spleen  is  swollen  and 
dark  in  color,  as  also  is  the  liver,  which  shows  much  focal  necrosis. 
The  glycogen  content  of  the  liver  is  decreased  in  abrin  poisoning.  ^^ 
Subcutaneous  injection  causes  local  edematous  inflammation  without 
suppuration.  Histologically,  in  the  most  affected  organs  are  found 
much  cellular  necrosis  and  disintegration,  especially  of  lymphoid  and 
epithelial  cells.  Changes  in  the  capillary  endothelium,  fibrinous 
thrombi,  and  abundant  hemorrhagic  extravasations  are  widespread. 
Probably  agglutinative  thrombosis  by  red  corpuscles  plays  an  im- 
portant part  in  these  intoxications  (Ehrlich),  but  Aschof^-  ascribes 
the  thrombosis  to  the  fragments  of  disintegrated  marrow  and  blood 
cells.  The  great  amount  of  intestinal  injury  probably  depends  upon 
the  fact  that  these  poisons  are  largely  eliminated  through  the  intestinal 
mucosa.  There  are  also  severe  changes  in  the  bone  marrow,  accom- 
panied  by  the  appearance  of  micleated  (M ytlnocytes  in  the  blood. ^•■' 

Mushroom  Poisons.'^ — The  jjoisons  of  the  three  chief  poisonou.s  imishrooms, 
Avinnitd  viuscari'i,  IlcivcUa  escitlrnta,  and  Amnnita  ph  nil  aides,  differ  from  one 
another  quite  essentially.  The  poisonous  principles  of  tiie  first  and  second, 
muscarine  and  helvellic  acid,  are  non-protein  substances,  of  known  chemical  com- 

»  Jour.  Kxper.  Med.,  1897  (2),  197. 
'"  Amer.  Jour.  Med.  Sei.,  190.3  (12(>),  200. 
"  Doyon,  Coinpt.  Ifend.  Soc.  liiol.,  1909  (t)7),  ^50. 
'=  Arch.  Int.  Med.,  ]\i\A  (12),  iiOA. 
'•'  HuntiuK,  .Jour.  Kxper.  Med.,  190()  (8),  t)2'). 

'Miesume  l)y  Mtirner,  TTpsala  L:ikaref.  Korh..  I9I9  (21).  1.  l'atli(»l()Kical 
anatomy  described  I)y  TryMi,  xirchows  .\rehiv..  1919  (22(1),  229. 


SNAKE  VEXOMS  J  11 

position,  which  :iio  discussed  elsewhere;  hut  the  A7nanila  phalloides,  the  most 
important  of  tho  tiuoo,  owes  its  toxic  proijcrtics  to  at  least  two  poisonous  con- 
stituents. One  is  powerfully  hemolytic,  is  desiioyod  hy  heating  thirty  minutes  at 
0.')°,  and  acts  directly  upon  red  corpuscles  without  the  presence  of  serum. "^ 

The  studies  of  Ford'"  and  his  associates  have  shown  that  this  hemolysin  is  a 
glueoside,  j'ielding  on  hydrolysis  pentose  and  volatile  bases,  and  yet  capable  of 
actinfi  as  an  antigen,  since  actively  antihemolytic  sera  can  })e  produced  by  im- 
munizing animals.  This  substance  corresponds  to  the  pfinllin  of  Kobert.  Prolj- 
ably  this  hemolytic  poison  is  not  the  important  agent  in  poisoning  by  Amanita, 
as  it  is  easily  destroyed  by  heat  and  the  digestive  fluids.  The  thermostable  poi.son, 
A7/i(iuita-t(>.rin,  gives  no  reactions  for  either  glucosides  or  proteins,'^  and  does  not 
confer  any  considerable  antito.xic  jiroperty  on  the  blood  of  immunized  animals. 
The  toxin  kills  acutely,  the  animals  dj-ing  in  24 — 48  hours,  and  showing  no  changes 
beyond  a  fatty  degeneration  of  the  internal  organs.  The  hemolysin  kills  slowly 
in  3 — 10  days,  causing  local  edema  and  hemoglobinuria. 

Amanita  muscaria  contains  a  heat-resistant  agglutinin  which  also  seems  to  be  a 
glucoside,  but  it  is  not  toxic  nor  antigenic. 

An  extensive  study  of  many  fungi  by  Ford'*  led  him  to  classify  the  toxic  action 
in  three  groups:  (1)  nerve  poisons,  e.  g.,  muscarine;  (2)  those  causing  structural 
changes  in  the  viscera,  e.  g.,  A.  phallnidcs,  causing  fatty  degeneration;  (3)  gastro- 
intestinal irritants,  c.  g.,  Lactariits  torjuinosus. 

The  poison  of  Rhus  toxicodendron  has  also  been  found  bj'  Acree  and  Synie"  to 
be  a  glucoside,'-"  and  the  same  is  true  of  the  poison  oak,  Rhus  diversiloba,  which 
has  no  antigenic  properties. 2' 

(The  effects  of  the  phytotoxins  on  the  blood  are  discussed  under 
"Hemolysis"  in  Chapter  ix.  Vegetable  hemolytic  poisons  that  do 
not  resemble  the  toxins,  e.  g.,  glucosides,  etc.,  will  also  be  found  dis- 
cussed under  the  same  heading.) 

ZOOTOXINS" 

Snake  Venoms  -^ 

This  important  class  of  poisons,  first  thoroughly  investigated  by 
Weir  Mitchell  (I860),  and  Mitchell  and  Reichert  (1883),  has  re- 
cently aroused  great  interest  through  its  relations  to  bacterial  toxins 
and  the  problems  of  immunity.  The  poisons  of  different  species  of 
snakes  seem  to  have  much  in  common  with  one  another,  whether  de- 
rived from  the  Elaiperine  snakes  (cobras  and  numerous  other  Indian 
and  Australian  snakes),  or  Viperidce  (including  most  poisonous  Amer- 
ican snakes),  or  Hijdrophince  (the  poisonous  sea-snakes),  although 
very  characteristic  ditferences  exist  between  each. 

'*  The  hemagglutinin  of  Agaricus  cam,pestris  is  precipitated  at  a  H-ion  concen- 
tration of  2.G  X  10-^  (Brossa,  Arch.  sci.  med.,  1915  (39),  241). 

'6  See  Jour,  of  Pharm.,  1910  (2),  145;  1913  (4),  235,  241,  and  321. 

'^  Rabe  (Zeit.  exp.  Path.,  1911  (9),  352)  considers  it  to  be  an  alkaloid. 

'»  Jour,  of  Pharm.,  1911  (2),  285. 

'9  Jour.  Biol.  Cheni.,  1907  (2),  547. 

-"Questioned  by  McNair,  Jour.  Amer.  Chem.  Soc,  1916  (38).  1417. 

^'  Adelung,  Arch.  Int.  Med.,  1913  (11),  148. 

--  Full  review  and  literature  given  by  Faust,  "Die  tieris.ihen  Gifte,"  Braun- 
schweig, 1906;  also  in  Abderhalden's  Handbuch,  Vol.  II.  iSachs,  Kolle  and  Was- 
sermann's  Handbuch,  1913  (2),  1407. 

-^  Elaborate  review  and  bii)Iiography  given  by  Noguchi,  Carnegie  Institution 
Publications,  1909  No.  Ill;  also  by  Calmette,  "Les  venins.  les  animaux  venimaux 
et  la  serotherapie  antivenimeuse,"  Paris,  Masson,  1907;  Calmette.  Kolle  and  "VVas- 
sermann's  Handbuch,  Vol.  II,  p.  1381;  with  reference  to  North  American  snakes, 
see  Prentiss  Wilson,  Arch.  Int.  Aled.,  1908  (1),  516. 


142  PHYTOTOXINS  AND  ZOOTOXINS 

The  essential  anatomical  differences  between  the  different  classes  of  snakes  are 
as  follows:  Colubrickp,  whicli  include  all  the  non-poisonous  snakes,  have  no 
mechanism  for  injecting  poisons  into  their  victims.  Colubridce  venenoscB  are 
venomous  snakes  resembling  in  many  particulars  the  harmless  Colubrines.  but 
having  short  poison  fangs,  firmly  fastened  to  the  maxilla  in  an  erect  position; 
in  this  class  are  included  the  cobra  and  the  venomous  snakes  of  Australia.  Vi- 
peridce,  or  vipers,  are  characterized  by  a  highly  specialized  apparatus  for  in- 
jecting the  poison;  their  poison  fangs  are  very  long,  and  the  maxillary  bone,  to 
which  they  are  fastened,  is  so  articulated  that  it  rotates  about  a  quarter  of  a 
circle  when  the  snake  strikes,  bringing  the  fangs  into  an  erect  position.  The 
fangs  are  canalized  and  pointed  at  the  end  like  a  hypodermic  needle,  and  the 
poison  is  forced  through  them  under  considerable  pressure  bj^  a  large  muscle  that 
contracts  over  the  salivary  gland.  Accessory  fangs  in  various  stages  of  develop- 
ment are  also  present  to  replace  any  fang  lost  in  action.  All  the  poisonous  snakes 
of  Morth  America,  with  one  insignificant  exception,  belong  to  the  vipers,  and  to 
a  special  class  known  as  the  "pit  vipers,"  because  of  the  presence  of  a  deep  pit 
of  unknown  function  above  the  maxilla.  The  exception  mentioned  is  the  "coral 
snake"  found  on  the  coast  of  Florida,  around  the  Gulf  of  Mexico  and  in  the  south- 
eastern states;  it  is  a  member  of  the  colubrine  poisonous  snakes,  of  small  size,  and 
seldom  causes  serious  poisoning.  The  poisonous  vipers  are  the  rattlesnakes 
(Crotahis),  of  which  there  are  some  ten  to  twelve  or  more  species,  and  Sistrurus  of 
which  there  are  two  species ;  the  copperhead  adder  {Andstrodon  coniroirix)  and 
the  water  mocassin  {Andstrodon  piscivorus). 

The  classification  used  above  is  the  one  followed  in  most  publications  on 
poisonous  snakes;  a  more  modern  classification  divides  the  snakes  {Ophidia)  into 
several  .series,  one  of  these  including  all  poisonous  snakes  under  the  title  of  Pro- 
teroglypha,  and  dividing  this  series  into  the  three  families:  (1)  Elapimv,  including 
cobras,  coral  snakes,  etc.;  (2)  Hydrophince,  the  poisonous  sea-snakes;  (3)  Viperidce, 
including  all  snakes  with  erectile  fangs. -^ 

The  source  of  the  venom  is  probably  in  part  the  blood,  since  snake 
blood  has  been  found  to  contain  poisons  very  similar  to  some  of  those 
in  the  venom;  therefore  these  are  presumably  simplj^  filtered  out  by 
the  venom  glands,  and  not  manufactured  by  them.^^  Other  poison- 
ous constituents  of  venom  are  not  found  in  snake  serum,  and  there- 
fore are  probably  manufactured  by  the  venom  gland.  Apparently 
many  of  the  harmless  snakes  produce  a  poisonous  saliva,  since  extracts 
of  their  glands  are  said  by  Blanchard^^  to  possess  the  properties  of  the 
venoms,  and  if  so  these  snakes  are  harmless  chiefly  because  they 
lack  an  apparatus  for  injecting  the  poison.  As  a  rule,  however,  the 
venom  glands  are  much  more  highly  developed  in  the  poisonous  snakes, 
and  are  connected  with  a  specialized  injection  apparatus;  in  structure 
they  are  compound  racemose  glands. 

Properties  of  Venom. — As  ejected,  the  venom  is  weakly  acid  or 
neutral  in  reaction,  and  free  from  bacteria,  contrary  to  earlier  ideas 
(Langmann).  Its  specific  gravity  is  1030  to  1077,  and  it  contains  a 
large  amount  of  solids,  generally  20  to  40  per  cent,  by  weight.  These 
are  precipitated  by  alcohol,  ether,  tannin,  and  iodin,  but  do  not  ad- 

^*  For  a  full  discussion  of  the  characteristics  of  the  poisonous  snakes  of  North 
America,  see  the  monograph  with  that  title  by  Stejneger,  Report  of  U.  S.  Na- 
tional IVIuseum,  1S<.).'{,  Wasliingtou.  A  good  summary  is  also  given  by  Langmann, 
Jlefcreiic(!  Handbook  of  Medical  Sciences,  ('oiicerniiig  })oisonous  sca-snakos, 
Ili/droplniliu,  see  Hoiilanger,  Natural  Science,  1S92  (1),  41.  The  poisonous  snakes 
of  India  are  descril)ed  by  Fayrer,  in  "The  Thanatopliidia  of  India,"  JiOndon,  1874. 

'^''  Contradicted  by  Arthus,  Arch,  internat.  phvsiol,  1912  (12),  102. 

^»  Com])t.  Uend.  Soc.  Biol.,  1894  (40),  35. 


SNAKE  VENOMS  143 

here  to  precipitates  of  phosphates  as  do  enzymes  and  toxins  (Cal- 
mette).  They  do  not  diffuse  through  dialyzing  membranes.  When 
dried,  the  venom  can  be  kept  almost  indefinitely  without  losing  its 
strength,  specimens  over  twenty  years  old  having  been  found  unim- 
paired. Glycerol  and  alcohol  also  seem  not  to  injure  it,  but  oxidiz- 
ing agents  of  all  kinds  arc  very  destructive.  Light  impairs  the  power 
of  venoms,  as  also  does  radium  (Phisalix).^^  Eosin  and  erythrosin 
also  reduce  the  power  of  venom  through  their  photodynamic  action, 
affecting  the  neurotoxic  properties  less  than  the  hcmatotoxic  compo- 
nents (Noguchi).^^  Cobra  venom  withstands  even  100°  for  a  short 
time,  but  crotaline  venoms  are  destroyed  at  80-85°. 

Much  work  has  been  done  upon  the  nature  of  the  constituents  of 
venom.  As  early  as  1843  Prince  Lucien  Bonaparte  found  that  there 
were  proteins  in  the  venom,  which  was  corroborated  by  Mitchell  in 
1861.  In  1883  Mitchell  and  Reichert  described  two  poisonous  pro- 
tein constituents  of  venom,  one  of  which  was  coagulable  by  heat  and 
seemed  to  be  a  globulin;  the  other  resembled  the  proteoses  (they 
called  it  "peptone,"  according  to  the  nomenclature  of  that  time). 
To  the  globulin  they  ascribed  the  local,  irritating  properties  of  venom  • 
to  the  albumose,  the  systemic  intoxication.  Corresponding  to  their 
action,  venoms  of  different  serpents  were  found  to  vary  greatly  in  the 
proportions  of  these  proteins.  Cobra  venom,  which  acts  chiefly  sys- 
temically,  contains  98  per  cent,  of  albumose  and  but  2  per  cent,  of 
globulin;  rattlesnake  venom,  with  its  marked  local  effects,  contains 
25  per  cent,  of  the  irritating  globulin;  moccasin  venom  contains  8 
per  cent,  of  globulin.  Several  other  observers  soon  corroborated  the 
main  facts  of  Mitchell  and  Reichert's  report;  but,  as  has  been  seen  in 
connection  with  the  consideration  of  the  composition  of  enzymes,  tox- 
ins, etc.,  the  fact  that  a  substance  is  carried  down  with  a  protein  is 
no  proof  that  it  is  itself  a  protein.  What  has  been  established  is 
merely  that  the  irritating  component  of  venom  can  be  destroyed  by 
heat,  and  is  removed  with  the  globulin  in  fractional  separation;  while 
there  remains  a  substance  not  destroyed  by  boihng,  which  comes  down 
at  least  in  part  with  the  albumoses  of  the  venom,  and  causes  chiefly 
systemic  manifestations. 

Since  venoms  act  as  antigens  and  stimulate  the  formation  of  spe- 
cific antibodies,  it  is  to  be  presumed  that  the  poisonous  principles 
are  proteins,  or  toxalbumins,  although  this  conclusion  does  not  neces- 
sarily follow.  Faust^^  believes  the  poison  of  venoms  not  to  be  proteins, 
but  glucosides,  free  from  nitrogen,  resembling  very  much  quillajic 
acid,  and  therefore  belonging  to  the  saponin  group  of  hemolytic 
agents.  He  has  isolated  such  a  substance  from  cobra  venom,  which 
he  calls  ophiotoxin  (C17H26O10),  and  from  rattlesnake  venom  a  sub- 

"  Compt.  Rend.  Soc.  Biol.,  1904  (56),  327. 

=«  Jour.  Exper.  Med.,  1906  (8),  252. 

-9  Arch.  exp.  Path.  u.  Pharni.,  1907  (56),  236;  19111(64),  244 


144  PHYTOTOXINS  AND  ZOOTOXINS 

stance  which  seems  to  be  a  polymer  of  the  ophiotoxin,  (C34H54O21). 
Possibly  these  glucosides  are  bound  to  proteins,  forming  compound 
proteins  which  act  as  specific  antigens.  According  to  this  work  the 
snake  venoms  and  the  dermal  poisons  of  toads  and  frogs  are  all  closely 
related  substances. 

Enzymes  in  Venoms. — .\s  venom  causes  rapid  liquefaction  of  tissues  into 
which  it  is  injected,  Ploxner  and  Noguchi^"  tested  crotalus  and  cobra  venom  for 
proteases,  and  found  that  the\^  digested  muscle  rapidlj',  and  also  gelatin  and 
unboiled  fibrin;  whereas  boiled  fibiin  and  boiled  egg-albumen  were  undigested.^' 
Kinases  and  nucleases  are  also  present  in  venoms  (Delezenne).*-  \\'ehrmann'^ 
found  that  venom  digests  fibrin  and  inverts  saccharose,  but  does  not  digest  starch. 
Martin^^  found  fibrin  ferments  in  various  venoms,  which  are  probably  important 
agents  in  causing  thrombosis.  There  are  also  active  lipases  in  venom=,  to  which 
many  of  the  effects,  especially  hemolj'sis  and  fatty  degeneration  of  the  tissues, 
may  be  at  least  partly  due  (Noguchi),  and  the  hemolysin  of  cobia  venom  seems  to 
be  a  lipase  that  splits  lecithin  into  hemolytic  substances  (Coca).^*  Delezenne'- 
found  zinc  always  present  in  venom  and  attributes  to  it  a  relation  to  the  enzyme 
activity. 

Toxicity. — Calmette  has  determined  the  toxicity'  of  several  ven- 
oms, and  gives  the  following  figures: 

1  gm.  cobra  or  aspis  kills 4000  kgm.  of  rabbit. 

1  gm.  hoplocephaius  kills 3450  kgm.  of  rabbit. 

1  gm.  fer  de  lance  or  pseudechis  kills 800  kgm.  of  rabbit. 

1  gm.  Crotalus  horrid  us  kills 600  kgm.  of  rabbit. 

1  gm.  Pelias  berus  kills 250  kgrn.  of  rabbit. 

The  danger  of  the  bite  depends  not  only  upon  the  difference  in 
the  strength  of  the  venom  of  different  varieties  of  serpents,  but  also 
upon  the  size  of  the  snake,  the  time  of  year  and  condition  of  hunger  or 
plenty,  and  particularly  whether  the  entire  discharge  is  injected  suc- 
cessfully or  not.  The  fatal  dose  of  cobra  venom  for  an  adult  man  is 
variously  estimated  at  from  0.01  to  0.03  gm.,  while  the  venom  of 
Hydrophince  is  about  ten  times  as  toxic;  for  crotalus  venom  the  lethal 
dose  is  probably  0.15  to  0.3  gm.  (Noguchi).  Probably  in  the  major- 
ity of  strikes,  by  no  means  all  the  fluid  ejected  by  both  fangs  is  in- 
jected beneath  the  skin  of  the  victim.  A  large  diamond  rattler  may 
eject  as  much  as  a  half  teaspoonful  of  venom  at  one  discharge  and 
such  a  dose  would  usuallj'  l)e  fatal.  Repeated  ejections  decrease  the 
strength  of  the  venom  rapidly,  until  it  may  have  almost  no  toxicity. 
In  general,  venom  is  most  active  in  warm  weather  and  immediately 
after  the  snake  has  fed;  in  winter  its  toxicity  is  slight. 

The  mortality  in  America  from  snake-l)ites  is  very  hard  to  ascer- 
tain, various  authors  giving  figures  at  w'ulo  variance.     The  extensive 

»«  Univ.  of  i'uuM.  iMed.  liull.,  1902  (15),  300. 

'•  See  also  lloussay  and  Xegrete,  Revista  de  I'insl.  l)act.  Buenos  Aires,  1918  (1), 
431. 

32  Anil.  lust.  Paslcur,  1919  (33),  ()8. 
"  Ann.  (1.  I'ln.st.  I'ustcur,  1,S9S  (12),  510. 
^*  Jour,  of  IMiysiol.,  1905  (32),  207. 
^''Jour.  Infect.  Dis.,  1915  (17),  351. 


SNAKE  VENOMS  1  »•') 

studies  of  Willsou'"'  show  about.  t(Mi  per  cent .  mortality  from  all  venom- 
ous snakc-bitcs  in  this  country,  the  (Uffercnt  species  giving  figures  as 
follows:  Coral  snakes,  twenty  to  fifty  per  cent.;  water  moccasins, 
seventeen  per  cent.;  large  rattlesnakes,  eleven  to  twelve  per  cent.; 
copperheads  and  ground  rattlers,  no  mortality  excej)!  in  children  or  in 
cases  of  complications.  The  mortality  in  children  is  at  least  double 
that  in  adults.  Many  deaths  from  snake-bites  of  all  kinds  are  due 
to  the  treatment  rather  than  to  the  bite.  The  poisonous  snakes  of 
Australia,  although  numerous,  are  not  very  virulent,  and  the  mortality 
is  given  as  about  seven  per  cent.  A  full  charge  of  venom  from  the 
cobra  and  many  other  Indian  snakes  is  inevitably  fatal  (Fayrer). 
The  crotaline  snakes  of  the  tropics  are  more  venomous  than  those  of 
the  north,  Lacheris  laticeolatus  of  Central  America  and  Mexico  being 
nearly  as  dangerous  as  the  cobra. 

When  venom  is  taken  into  the  stomach  in  the  intervals  of  diges- 
tion, enough  may  be  absorbed  to  produce  death,  especially  in  the  case 
of  those  venoms  which  contain  a  large  proportion  of  the  albumose, 
which  is  dialyzable;  but  during  active  digestion  the  venom  undergoes 
alteration  and  is  rendered  harmless.  It  has  been  found  experiment- 
ally in  animals  that  cobra  venom  placed  in  the  stomach  causes  ordi- 
narily no  harm  whatever,  but  if  a  loop  of  the  intestine  is  isolated,  a 
fistula  established  and  allowed  to  heal,  venom  introduced  through 
this  opening  always  produces  death.  It  is  probably  not  so  much 
the  pepsin  and  hydrochloric  acid  that  destroys  the  venom,  as  the 
trypsin.  If  the  bile-duct  is  ligated,  the  venom  is  destroyed  just 
the  same.  Much  of  the  venom  seems  to  be  eliminated  into  the  stom- 
ach, no  matter  how  it  is  introduced  into  the  system,  and  apparently 
it  is  also  partly  excreted  by  the  kidneys.  Rattlesnake  venom  seems 
not  to  be  absorbed  through  mucous  membranes. 

Physiological  Action. — As  indicated  in  the  preceding  paragraph, 
the  effects  of  the  bites  of  different  classes  of  snakes  arc  quite  differ- 
ent.    Langmann  describes  the  symptoms  as  follows: 

Cobra  Poisoning. — "Within  an  liour,  on  an  average,  the  first  constitutional 
symptoms  ai)pear:  a  pronounced  vertigo,  quickly  followed  by  weakness  of  the 
legs,  which  is  increased  to  paraplegia,  ptosis,  falling  of  the  jaw  with  paralysis 
of  the  tongue  and  epiglottis;  at  the  same  time  there  exists  an  inability  to  speak 
and  swallow,  with  fully  preserved  sensorium.  The  symptoms  thus  resemble  those 
of  an  acute  bulbar  paralysis.  The  pulse  is  of  moderate  strength  until  a  few 
minutes  after  the  cessation  of  respiration;  the  latter  becomes  slower,  labored, 
and  more  and  more  superficial  until  it  dies  out  almost  impe.ceptibly.  Death 
occurs  at  the  latest  within  fifteen  hours;  in  32  per  cent,  of  all  cases  in  three  hours. 
There  are  very  few  local  changes."  Cushny"  finds  that  cobra  venom  produces 
paralysis  of  the  motor  nerve  terminations  of  .muscle,  resembling  the  action  of 
curare;  the  central  nervous  system  is  not  directlj^  involved.  Death  recults  from 
failure  of  the  moto  ■  nerve  ends  in  the  respiratory  muscles  to  transmit  impulses 
to  the  muscles.  Alkaloids  that  are  antagonistic  to  curare  (physostigmine,  guani- 
dine)  are  not  effective  in  cobra  poisoning,  but  are  them,selves  rendered  inactive. 

"«  Arch.  Int.  Med.,  1908  (1),  516. 

"  Trans.  Roy.  Soc,  London  (B),  191(5  (208),  1. 

10 


146  PHYTOTOXINS  AND  ZOOTOXINS 

Viper  Poisoning. — "After  the  bite  of  a  viper  the  local  changes  are  mo>t  pro- 
nounced; there  are  violent  pains  in  the  bleeding  wound,  hemorrhagic  discolora- 
tion of  its  surroundings,  bloody  exudations  on  all  the  mucous  membranes,  and 
hemoglobinuria.  Usually  somewhat  later  than  in  cobra  poisoning  constitutional 
symptoms  develop;  viz.,  great  prostration  with  nausea  and  vomiting,  blood  pres- 
sure falls  continuously,  and  respiration  grows  slow  and  stertorous.  After  a  tem- 
porar}'-  increase  in  reflexes,  paresis  supervenes,  with  paraplegia  of  the  lower 
extremities,  extending  in  an  upward  direction  and  ending  in  a  complete  paralysis. 
It  therefore  resembles  an  acute  ascending  spinal  paralysis.  If  the  patient  re- 
covers from  the  paralysi-s,  a  septic  fever  may  develop;  not  rarely  there  remain 
suppurating  gangrenous  wounds,  which  heal  poorl}^" 

It  will  be  noticed  that  there  is  lacking  the  usual  period  of  incuba- 
tion that  follows  injection  of  bacterial  toxins,  and  if  it  happens  that 
the  venom  has  been  injected  directly  into  one  of  the  veins,  death  may 
occur  within  a  few  minutes.  When  recovery  occurs,  the  disappear- 
ance of  symptoms  is  remarkably  abrupt,  within  a  few  hours  a  des- 
perately sick  person  becoming  almost  entirely  free  from  all  evidences 
of  the  intoxication. 

Pathological  Anatomy. — Postmortem  examination  shows  changes  varying  with 
the  nature  of  the  poisonous  snake  that  has  caused  death.  In  the  case  of  a  cobra 
bite,  according  to  Martin,  the  areolar  tissue  about  the  wound  is  infiltrated  with 
pinkish  fluid;  the  blood  is  often  fluid;  the  veins  of  the  pia  are  congested,  and 
the  ventricles  often  contain  turbid  fluid;  the  kidneys  may  show  much  congestion. 
When  death  occurs  in  a  few  minutes,  enormous  general  intravascular  clotting  is 
found,  which  seems  to  be  the  cause  of  death.  After  death  from  a  viper  bite  the 
site  of  the  wound  is  the  seat  of  intense  edema  and  extrava-ation  of  blood;  if  in 
the  muscles,  these  are  much  softened  and  disorganized.  Hemorrhages  are  found 
in  all  organs  and  in  the  intestinal  tract.  If  death  occurs  after  several  days  it 
is  generally  because  of  sepsis,  and  shows  the  usual  changes  of  this  condition;  in 
addition,  as  a  rule,  to  marked  gangrenous,  ulcerative,  and  sloughing  processes  at 
the  site  of  the  bite. 

Histologically  there  are  found,  in  addition  to  innumerable  hemorrhages  in  nearly 
all  the  organs,  many  vessels  plugged  with  thrombi  composed  of  more  or  less 
hemolyzed,  agglutinated  erythrocytes.  The  changes  produced  in  the  nervous 
tissue  by  the  Australian  tiger  snake  are  described  by  Kilvington,'*  who  found  marked 
chromatolysis,  the  Xissl  bodies  breaking  into  dust-like  particles,  and  eventually 
all  stainable  substance  disappearing  from  tlie  cytoplasm;  the  nucleus  retains  its 
central  7)osition,  but  often  loses  its  outline  and  may  disappear.  The  cells  around 
the  central  canal  of  the  cord  are  most  affected.  There  are  no  inflammatory 
changes  in  the  nervous  sj'stem,  and  if  death  occurs  very  quickly  there  may  be  no 
microscopic  alterations.  Hunter'^  found  similar  changes  in  the  Xissl  bodies  in 
both  krait  and  cobra  poisoning;  in  the  mcdullated  fibers  he  found  the  myelin 
sheath  converted  into  ordinary  fat.  The  venom  of  sea  snakes  {Enhydrina  vn'aka- 
dien)  has  a  severe  action  on  the  nervous  tissues,  while  Dnboia  has  none  (Lamb 
and  Hunter^").  Nowak"  studied  experimental  animals,  and  found  much  fatty 
change  in  the  livers,  even  if  death  occurrcil  one-half  hour  after  iH)isoning;  also 
focal  necrosis  in  the  liver,  acute  parenchymatous  alterations  in  the  kidney,  and 
pneumonic  patches  in  the  lungs. 

Effects  on  the  Blood. — There  has  been  much  discussion  concerning  the  part 
played  by  the  abundant  and  prominent  intravascular  clotting  in  causing  death 
after  snake-l)ite.  Lamb"  states  that  when  venoms  are  slowly  absorbed  tiie 
coagulahility  of  the  blood  is  decreased  and  it  is  found  fluid  after  death,  but  when 
a  fatal  do.se  of  venom  (vi!)er)  is  rapidly  absorbed,  clotting  is  increased  and  thmni- 
bosis  is  the  chief  cause  of  death.      Martin  has  demonstrated  very  lu-tivo  fibrin 

"  Jour,  of  Physiol.,  1902  (28),  426. 
"  Glasgow  Med.  Jour.,  190.'}  (59),  98. 
^"Lancet,  1907  (ii),  1017. 
*'  Ann.  d.  I'Inst.  I'asteur,  1898  (12),  3(59. 
"  Indian  Medical  Gazette,  Dec,  1901. 


SNAKK   VhWOMS  1  17 

forinciits  in  snake  venom  (Inc.  rit.).  It  is  highly  proliahl  ■,  iiowever,  thit  many 
of  tlie  thrombi  of  venom  poisoning  are  not  prixhiced  by  coagiihition  of  fibrin,  but 
by  agpihttination  of  tlie  r(>(i  corpusflos,  wliich  Floxncr'^  has  .sliown  can  cause 
largo  clots  in  the  heart  and  great  vessels,  as  well  as  "hyalin"  thrombi  in  the 
small  vessels,  lloussay""  states  that  most  snake  venoms  destroy  the  cytozyme 
(which  combines  with  serozyme  and  calcium  to  form  thrombin),  so  that  the  blood 
becomes  incoagulable.  The  Argentine  crotalus  and  lachesis  venoms,  however, 
coagulate  even  <'it  rated  blood. 

Nature  of  Venoms. — The  varied  effects  produced  by  venoms  have 
been  found  to  be  chic  to  a  number  of  poisonous  elements  which  they 
contain,  and  Avhich  have  been  chstinguished  and  separated  from  one 
another  by  Flexner  and  Noguchi.^"^  These  are  hemotoxins  [hemoly- 
sins and  hemagglutinins) ,  leucocytolysins,  neurotoxins,  and  endothel- 
iotoxins  {hemorrhagin) ,  but  it  must  be  taken  into  consideration  that 
Fausf*^  beheves  that  the  single  glucosidal  poison  which  he  has  found 
in  rattlesnake  venom  is  responsible  for  all  the  effects  of  the  venom, 
except  the  hemagglutination.  [In  another  place  (see  "Hemolysis") 
the  nature  of  the  hemolytic  agent  is  discussed.]  Venom  agglutinin  is 
quite  independent  of  the  hemolysin,  for  it  is  destroyed  by  heating  to 
75°-80°,  whereas  the  hemolysin  is  destroyed  only  partly  at  100°. 
Agglutinin  acts  in  the  absence  of  serum  complement,  and  therefore 
is  not  an  amboceptor;  it  is  apparentlj^  more  like  the  toxins  in  its  na- 
ture. The  agglutination  of  the  corpuscles  does  not  interfere  with 
their  subsequent  hemolysis.  Michel  states  that  the  agglutinin  of  cobra 
venom  can  be  separated  from  the  hemolysin  and  the  toxin  by  means  of 
ultrafiltration  through  collodion  membranes,  as  the  agglutinin  exists 
in  larger  molecular  aggregates. "^^  ' 

The  leucocytotoxins  were  found  by  Flexner  and  Noguchi  to  be 
quite  distinct  from  the  hemolysins,  for  after  saturating  all  the  hemoly- 
sin with  red  corpuscles,  the  venom  still  shows  its  effects  on  the 
leucocytes,  which  effects  consist  in  cessation  of  motility  and  disintegra- 
tion, affecting  particularly^  the  granular  cells.  The  leucocytotoxin, 
however,  resembles  the  hemolysin  in  that  it  appears  to  be  an  ambo- 
ceptor. Leucocytes  are  also  agglutinated  by  venom,  possibly  by  the 
same  agglutinin  that  acts  on  the  red  corpuscles.  Serum  complement 
is  inactivated  in  vitro  by  cobra  venom  through  changes  in  the 
globulins  brought  about  by  the  venoms.'*'^  By  saturating  venom  with 
either  red  corpuscles  or  nerve-cells  it  was  found  by  Flexner  and  No- 
guchi that  the  toxic  principle  for  each  is  distinct  and  separate. ^^ 
Other  sorts  of  cells,  however,  are  able  to  combine,  or  at  least  remove 
some  parts  of  the  toxic  elements,  but  to  a  much  less  degree.  The 
neurotoxin,  like  the  hemolysin,  resembles  an  amboceptor,  and  since 

*^  Univ.  of  Penn.  Med.  Bull.,  1902  (15),  324. 

""  Prensa  Med.  Argentina,  1919  (6),  133. 

*'  Jour.  Exp.  Med.,  1903  (9),  257;  l^niv.  Penn.  Med.  Bull..  1902  (15),  345. 

••s  Arch.  Exper.  Path.  u.  Pharm.,  1911  (64),  244. 

«  Compt.  Rend.  Soc.  Biol..  1916  (77),  150. 

"  Hirschfeld  and  Klinger,  Biochem.  Zeit.,  1915  (70),  398. 


148  PHYTOTOXINS  AND  ZOOTOXINS 

venom  contains  no  coniplonient,  the  neurotoxin  has  first  to  be  supplied 
with  complement  by  the  victim's  blood  or  tissues  before  it  can  harm 
the  cells.  The  venoms  are  not  only  toxic  for  mammalian  cells,  but 
also  for  simple  unicellular  organisms,  including;  bacteria;  tadpoles 
are  paralj'zed  in  solutions  containing  one  part  of  cobra  venom  per 
million.  ^'^ 

The  pronounced  hemorrhage-producing  property  of  serums,  par- 
ticularly that  of  the  rattlesnake,  was  also  found  to  be  due  to  a  specific 
toxin  acting  on  the  endothelium  of  the  capillaries  and  small  veins, 
and  not  to  the  changes  in  the  blood  itself,  as  had  formerly  been  thought. 
This  endotheliotoxin,  which  Flexner  and  Noguchi  call  "hemorrhagin," 
is  quite  distinct  from  the  other  toxic  substances,  being  destroyed  at 
75°,  a  temperature  that  leaves  the  neurotoxin  and  hemolysin  unin- 
jured. Its  endotheliolytic  action  is  show  in  the  glomerular  capil- 
laries, where  it  causes  hemorrhage  and  hematuria  (Pearce).^^ 

Variations  in  Venoms. — In  distribution  among  the  various  poi- 
sonous reptiles  these  toxins  seem  also  quite  distinct  from  one  another, 
which  explains  the  difference  in  the  effects  of  bites  of  snakes  of  various 
kinds.  Cobra  venom  contains  chiefly  neurotoxin,  hence  the  symp- 
toms of  cobra  bite  are  largely  of  nervous  origin,  with  but  little  local 
tissue  change.  Rattlesnake  venom  owes  its  effects  chiefly  to  hemor- 
rhagin, hence  the  marked  local  necrosis  and  extravasations  of  the 
blood,  and  the  generalized  hemorrhages;  the  nervous  effects  following- 
viper  bite  are  probably,  in  part,  due  to  hemorrhages  in  the  nervous 
tissue.  Cobra  venom  produces  great  hemolysis  and  little  agglutina- 
tion. Rattlesnake  venom  has  relatively  little  agglutinative  or  hemo- 
lytic power.  Water  moccasin  and  copperhead  venoms  are  more 
agglutinative  than  either,  and  intermediate  in  heniolytic  strength; 
they  cause  much  local  tissue  destruction. 

The  exact  action  of  cobra  venom  on  various  centers  and  organs  has  been 
studied  by  ElUot.^"  It  raises  blood  pressure  when  in  dihition  of  1:10.000,000. 
by  contractinfi;  vessels  and  stiinulatint;;  the  heart;  low  lethal  doses  kill  by  i)ara- 
lyzing  the  respiratory  center. 

Krait  (Bungarus  coeridues)  venom  acts  similarly,  but  loss  {xiwerfully,  and 
cannot  be  neutralized  by  Calmette's  antivenin.'^i 

Sea-snake  venoms  are  by  far  the  most  poisonous  of  all.  For  Enhi/drina  valaLd- 
dieu  the  lethal  dose  for  rabbits  is  O.OOOOti  ^ram  per  kilo  body  weight.  It  acts  by 
vagus  stimulation  and  paralysis  of  respiratory-  centers  ami  oi  motor  nerve- 
endings.''-' 

Hussell's  viper  {Ddhtda  Hussrllii)  owes  its  effects  chiefly  to  intravascular  dot- 
ting, according  to  Laiiil)  and  llauMa,'"'  and  conlnins  no  neurotoxin.  It  is  not 
neutralized  by  Calmette's  antivenin.  'I'iie  dots  are  ilue  to  agglutination  anil  con- 
tain no  fibrin  (Flexner). 

••*  Bang  and  Overton,  (Biochem.  Zeit.,  1911(31),  243)  state  that  corpuscles  can 
take  up  the  Mcurotoxin,  wliicli  is  s()lul)le  in  fats  and  lipoitls. 

^».)()ur.  Mxpcr.  iMcd.,  l'.U)'.>  (11),  .'):VJ. 

"Lancet,  l!»Ot  (i),  715. 

"'Elliot,  Siljjir,  and  ('ariiiidi:iel,  b:iiicft,   I'.KM  (ii),  IfJ. 

'- Fraser  and  J<:ili()t,  Lancet,  1004  (ii),  141;  a'so  Rogers,  .lour,  of  Phy.siol., 
1903  (;}0),  iv.  'I'lie  al)ove  are  also  given  completdv  in  the  Philosophical  'hans- 
actions  of  the  iloyal  Societv,  1904-,'),  vol.  1S7. 

'•'.Jour,  of  I'a'tli.  and  liact.,  1902  (S),  1. 


.S.V.I A' A'   VK.XOMS  14<) 

The  "(lila  Munst(M"  {Ilrlotlcrmn  suspect  urn)  sclddrii  (■.•iii.s(>s  .serious  i)()is()iiiiin 
ill  man,  l)ut  may  kill  small  animals,  suoli  as  frops.''  Its  poison  is  only  slightly 
iKMnolytic.  hut  prochicos  (Icucncnitivc  clianpcs  in  the  nervous  system  fLanRinann). 
The  liemohsin  is  activated  by  lecithin  (Cooke  and  Loeb).  An  elaborate  series  of 
studies  bv  Leo  Loeb  and  his  associates  give  all  the  known  facts  concerning  the 
Gila  Monster." 

Loss  of  Bactericidal  Powers. — The  frequency  of  inurketl  and 
persistent  sloughing  and  suppuration  at  the  site  of  snake-bites,  particu- 
larly from  the  vipers,  and  the  common  termination  in  sepsis,  was 
attributed  by  Welch  and  Ewing^^  to  a  loss  of  bactericidal  power  of 
the  blood,  which  they  found  followed  experimental  venom  poisoning. 
This  has  been  ascribed  by  Flexner  and  Noguchi  to  saturation  of  serum 
complement  by  the  numerous  amboceptors  of  the  venoms,  so  that  no 
complement  is  left  for  the  serum  to  use  against  the  bacteria.  In 
serum  whose  complements  do  not  combine  with  the  venom  ambocep- 
tors (e.  g.,  Necturus)  the  normal  bactericidal  powers  are  not  in  the  least 
impaired  by  the  addition  of  venom.  Morgenroth  and  Kaya  ascribe 
the  loss  of  complement  to  a  destruction  by  some  agent  in  the  venom. 

Snake  Serum. — The  serum  of  serpents  is  also  toxic  for  other  animals,"  even 
when  the  serpent  is  not  a  venomous  one;  c.  g.,  the  harmless  pine  snake  (Pitijophis 
cateniferis).  The  toxicity  of  snake  serum  seems  to  depend  chiefly  uj)on  its  hemo- 
toxic etTects  (hemagglutination  and  hemolysis),  the  toxic  substances  resembling 
amboceptors  and  similar  to,  but  not  altogether  identical  with,  the  amboceptor  of 
the  venoms.  Crotalus  tissues  also  produce  poisoning  in  proportion  to  the  blood 
they  contain,  but  are  without  toxic  effects  of  their  own  (Flexner  and  Noguchi). 

Antivenin. — Snake  venom  has  the  essential  property  of  all  true 
to.xins  of  immunizing,  with  the  appearance  of  an  antitoxin  in  the 
blood.  The  first  successful  immunizations  seem  to  have  been  made 
by  Sewall,^*  but  the  practical  production  of  antitoxic  serum  was  first 
accomplished  by  Calmette^^  and  by  Fraser.''°  At  first  it  was  be- 
lieved that  cobra'  antivenin  neutralizes  the  neurotoxins  and  hemoly- 
sins of  venoms  of  any  origin,  and  also  of  snake  serums,  and,  therefore, 
should  be  quite  effective  against  cobra  and  similar  venoms  which  pro- 
duce chief!}-  neurotoxic  and  hemolj^tic  changes.  This  implies  that 
these  toxic  substances  are  of  identical  nature  in  all  snakes,  no  matter 
how  dissimilar  the  snakes  may  be,  but  various  investigators,  especially 
Lamb,  have  found  sufficient  specificit}'  exhibited  by  different  venoms 
and  antivenoms  to  indicate  the  necessity  of  employing  the  specific 
antiserum  in  each  case  of  snake  bite.  A  special  antitoxin  against 
rattlesnake  venom  and  its  hemorrhagic  to.xin  has  been  successfully 

'*  Thorough  study  by  Van  Denburgh  and  Wright,  Amer.  Jour,  of  Physiol.. 
1900  (4),  209.  ' 

"  Carnegie  Inst.  Publication  No.  177,  1913. 

*«  Lancet,  1894  (1),  1236;  Ewing,  Med.  Record,  1894  (45),  663. 

°'  Questioned  by  Welker  and  Marshall,  Jour.  Pharmacol.,  1915  (6),  563. 

''  Jour,  of  Physiol.,  1887  (8),  203. 

"  Ann.  d.  I'lnst.  Pasteur,  1894  (6),  275:  also  subsequent  articles  in  1897  (11). 
214;  1898  (12),  343. 

««  British  Med.  Jour.,  1895  (i),  1309. 


150  PHYTOTOXINS  AND  ZOOTOXINS 

prepared  by  Noguclii.*''  This  crotalus  antivenin  also  neutralizes 
hemolysins  of  various  venoms,  and  also  of  snake  serums. 

Presumably  antivenin  neutralizes  venoms  in  exactly  the  same  way 
that  antitoxin  neutralizes  toxins;  i.  e.,  cell  receptors  are  thrown  off 
from  the  injured  cells  during  immunization,  which  combine  with 
venom  amboceptors  in  the  blood,  and  thus  prevent  their  combining 
with  the  cells.  Antivenin  also  prevents  the  inliibiting  action  of  venom 
on  bactericidal  serum,  indicating  that  it  prevents  the  venom  ambo- 
ceptors from  binding  the  serum  complement.  The  reaction  of  venom 
and  antivenin  is  certainly  a  chemical  one,  being  likened  bj'  Kyes^- 
to  that  of  strong  acids  upon  strong  bases. 

The  serum  of  animals  immunized  to  venoms  contains  precipitins 
for  the  proteins  of  these  venoms,  and,  to  some  extent,  for  the  serum 
proteins  of  the  same  species  of  snakes.  These  precipitins  are  highly 
but  not  absolutely  specific,  and  they  bear  no  exact  quantitative  rela- 
tion to  the  other  antibodies  present  in  the  same  sera.^^ 

As  is  well  known,  snakes  are  nearly  or  quite  insusceptible  to  snake 
venom.  Cunningham^*  found  that  serum  of  cobras  was  devoid  of 
antitoxic  property,  so  the  immunity  of  snakes  must  be  ascribed  to  an 
absence  of  cell  receptors  in  their  tissues,  with  which  their  venom  am- 
boceptor can  combine.  The  reputed  immunity  of  the  mongoose  and 
hedgehog  depends  partly  on  a  relatively  low  susceptibility,  but  prob- 
ably more  on  the  agility  of  the  mongoose  and  the  defensive  spines  of 
the  hedgehog. 

Platypus  Venom. — The  only  mammal  with  a  venomous  secretion  is  that  strange 
freak,  the  duck-billed  platypus  {Omithorhynchus  paradoxus).  The  males  have  a 
hollow  movable  spur  on  each  hind  foot,  communicating  like  a  fang  with  the  venom 
gland,  which  secretes  a  venom  with  properties  resembling  the  venoms  of  the 
Australian  snakes,  but  much  weaker. 

Scorpion  Poison  " 

This  poison  is  secreted  by  a  pair  of  specialized  glands  in  the  pos- 
terior segment  of  the  elongated  abdomen,  surrounded  by  a  firm  cap- 
sule with  a  sharp  apex  through  which  the  poison  is  discharged.  Its 
effect  on  man  is  usually  confined  to  local  pain,  swelling,  and  occa- 
sionally phlegmonous  inflammation  with  constitutional  symptoms 
after  bites  from  the  largest  species.  In  Africa  a  large  scorpion  {An- 
drocionus)  exists,  that  is  reputed  frequently  to  cause  fatal  poisoning, 
especially  in  children.  Manchuriaii  scorpions  {Buihus  martcnsi, 
Karchi)  seem  to  be  less  toxic  than  this  or  Mexican  scorpions  {Centi'urus 

«i  Univ.  of  Penn.  Med.  Bull,  1901  (17),  154;  Jour.  Expcr.  Med.,  1906  (8),  614. 

«-  liorl.  kliii.  Woch.,  190  t  (11),  494. 

*^  See  lloussay  and  Negrcte,  Hev.  inst.  bact.  Buenos  Aires,  1918  (1),  15. 

«'  Nature,  1S9()  (55),  139. 

*"*  A  comi)l('to  discussion  of  the  literature  on  i)ois()nous  invertebrates,  etc.,  is 
given  by  v.  Fiirth,  "  N'crglcicliende  chemische  IMiysioldgic  dcr  niedcren  Tiere," 
Jena,  190.'};  and  by  Faust,  "Die  (icrisclicn  (Jifte,"  liraunschweig,  190().  Con- 
cerning scorjjions  see  Kubota,  .Jour.   I'li.irriiacol.,   191S  (11),  447. 


SCORPION  POISON  151 

exlicauda,  Wood).  In  Korea,  however,  of  81  cases  collected  by  Mori,'^ 
four  were  fatal.  The  majority  of  serious  results  following  scorpion 
bites,  as  well  as  bites  of  poisonous  insects  to  be  considered  later,  are, 
however,  due  to  infection  of  the  wound,  which  occurs  readily  because 
of  local  necrosis  and  hemorrhages,  and  also  because  of  the  unfavorable 
conditions  existing  in  tropical  climates.  Apparently  these  bites  favor 
local  infection  much  as  do  those  of  vipers. 

When  general  symptoms  do  occur,  they  are  described  as  resembling 
strychnine  poisoning,  with  trismus,  stiffness  of  the  neck  and  eventu- 
ally of  the  respiratory  muscles,  which  seems  to  be  the  chief  cause  of 
death  (Cavorez).  Thompson,"  however,  observed  only  seldom  severe 
symptoms,  consisting  of  general  paralysis  that  passed  off  in  a  few 
hours.  Most  experimenters  with  scorpion  poison  describe  it  as  chiefly 
a  nerve-tissue  poison,  and  it  also  seems  to  act  as  a  hemolysin  and  ag- 
glutinin (Bellesme  and  Sanarelli),  but  Todd*^^  found  it  without  ac- 
tion on  corpuscles  and  not  capable  of  combining  with  nervous  tissues. 
Houssay^^  states  that  scorpion  poison  (B.  quinquestriatus)  is  above 
all  a  muscular  poison  of  the  veratrine  type,  and  a  powerful  peripheral 
excitant  of  the  salivary  and  lachrymal  secretions. 

Calmette^°  gives  the  lethal  dose  for  a  guinea-pig  as  0.5  milligram, 
while  Phisalix  and  Varigny  put  it  at  0.1  milhgram  and  state  that 
scorpion  blood  is  also  poisonous.  Wilson^ ^  found  the  toxicity  of  the 
venom  equal  to  0.1  gram  per  million,  that  is,  one  gram  of  poison  will 
kill  10,000,000  grams  of  guinea-pig, ^^  hence  it  is  much  stronger  than 
cobra  venom.  It  is  quite  stable,  and  keeps  many  months  in  an  ice 
chest;  is  not  affected  by  heating  to  100°  for  a  brief  period,  but  is  de- 
stroyed after  12  or  13  minutes'  heating.  The  active  constituents  are 
precipitated  by  saturating  with  ammonium  sulphate,  or  by  an  excess 
of  alcohol.  They  are  destroyed  by  either  pepsin  or  trypsin  (Kubota).^^ 
The  average  amount  of  toxin  in  an  Egyptian  scorpion  {Buthus  quin- 
questriatus) is  sufficient  to  kill  about  35  kilos,  which  agrees  with  the 
fact  that  fatal  poisoning  by  this  scorpion  is  rare  in  adults,  but  reaches 
60  per  cent,  in  children.  The  venom  is  harmless  when  taken  into  the 
stomach,  and  is  said  to  be  made  inactive  by  ammonia,  calcium  hypo- 
chlorite, and  peroxide  of  hydrogen.  Calmette  claims  that  antivenin 
for  cobra  in  part  neutralizes  scorpion  poison,  a  statement  which  could 
not  be  corroborated  by  Todd,  who  succeeded,  however,  in  preparing 
an  efficient  antiserum  by  immunizing  horses  with  scorpion  venom. ^^ 

5^  Korean  Med.  Soc.  Jour.  (Chosen  Igakukai-Zasshi),  1917,  p.  47. 

"  Proc.  Acad.  Nat.  Sci.  of  Philadelphia,  1886,  p.  299. 

«8  Jour,  of  Hygiene,  1909  (9),  69. 

"Jour,  phvsiol.  path,  gon.,  1919  (18),  305. 

'">  Ann.  Inst.  Pasteur,  1895  (9),  232. 

''  Records  of  Egyptian  Gov't.,  School  of  Med.,  1904;  abst.  in  Jour,  of  Phvsiol., 
1904  (31),  p.  xlviii. 

'^  Exactl}^  the  same  toxicity  is  shown  by  Korean  scorpions  (Mori).''^ 

"  A  successful  serum  has  also  been  prepared  in  Brazil  (see  Brazil-Medico, 
1918  (32),  161). 


152  PHYTOTOXINS  AND  ZOOTOXINS 

Houssay^*  also  describes  the  antiscorpion  serum  as  strictly  specific. 
A  large  number  of  naturalists  and  raconteurs  have  furnished  interest- 
ing tales  of  suicide  by  scorpions,  which  are  more  than  improbable  in 
the  light  of  our  present  knowledge  concerning  natural  immunity. 
Many  animals  seem  to  possess  more  or  less  immunity  to  scorpions 
(Wilson) J  especially  such  wild  animals  as  are  much  exposed  to  them. 

Spider  Poison 

The  poison  apparatus  of  the  spiders  consists  of  two  long  pouches 
lying  in  the  thorax  and  extending  into  the  jaws,  at  the  apex  of  which 
the  poison  is  discharged.  Some  of  the  larger  members  of  the  family 
are  very  poisonous,  e.  g.,  the  Malmignatte  (Latrodectes  tredecim- 
guttatas),  of  the  vicinity  of  the  lower  Volga  in  southern  Russia,  is 
said  to  have  destroyed  70,000  cattle  in  one  year,  the  bite  being  fatal 
in  12  per  cent,  of  all  cases,  although  rarely  killing  man.  Other 
members  of  this  species  in  Chili,  Madagascar,  and  other  countries 
are  not  much  less  venomous.  Kobert^'*  has  studied  the  poison  of  Mal- 
mignatte and  found  it  distributed  throughout  the  body  of  the  spider, 
even  in  the  eggs,  and  resembling  in  nature  the  snake  venoms.  It  is 
destroyed  by  heating,  and  seems  to  be  of  protein  nature;  the  chief 
effect  is  upon  the  nervous  system  and  heart. "^ 

A  number  of  common  spiders  investigated  by  Kobert  were  ap- 
parently not  poisonous  for  mammals,  except  the  "cross  spider"  (Epe- 
ira  diadema) ,  which  has  since  been  thoroughly  studied  by  him  and  by 
Sachs.''**  Walbum"  states  that  the  chief  poison  of  these  spiders  is 
found  in  the  ovaries,  the  salivary  poison  being  much  weaker,  and  the 
hemolysin  is  found  chiefly  in  the  albumin  fraction.  Epeiratoxin  re- 
sembles the  snake  venoms  strikingly,  according  to  Sachs,  for  it  con- 
tains a  powerful  hemolysin  which  he  calls  "arachnolysin,"  acting  very 
differently  with  different  sorts  of  blood,  and  destroyed  by  heating  at 
70°-72°  for  forty  minutes,  and  it  behaves  with  lecithin  and  cholesterol 
like  cobra  venom. ^^  By  imnmnizing  a  guinea-pig  Sachs  succeeded  in 
securing  an  antitoxin  of  some  strength.  The  agglutinin  is  quite  dis- 
tinct from  the  hemolysin. ^^  Only  such  blood  is  hemolyzed  as  is  able 
to  bind  the  poison  in  the  stroma  of  the  red  corpuscles.  The  discovery 
of  this  hemolysin  explains  Robert's  observation  of  hemoglobin, 
methemoglobin,  etc.,  in  the  urine  of  persons  bitten  by  spiders.  Sjiider 
hemolysins  have  been  studied  extensively  by  Houssay,**"  who  finds 

'^  "BeitrJige  zur  Kentnisse  der  Giftspinnen,"  Stuttgart.  1901. 

"•  In  western  America  and  South  America  is  foiind  a  sj>ider  {Latrodectes  uiae- 
lans)  the  bite  of  wliich  is  capable  of  causing  severe  spasm  of  the  ah(h)minal  musch\s. 
according  Ui  At  wood  (Southern  Californ.  Pract.,  \'ols.  10,  12  and  10).  Kellogg 
and  (4)lcman  (.lour,  of  Para>itoI.,  1915  (1),  107),  found  extracts  of  the  i)oison 
glands  of  this  spider  to  he  iiitililv  toxic. 

'«  Ilofmeister's  Heitr..  1902  (2),  125. 

"  Zeit.  immunitiit.,  1915  (2:5),  02."^. 

'*Pini,  1!  I'oliciinico  (Se/,.  Med.),  1909  (10),  20S. 

'"v.  S/.ily.  Zeit.  Immunitat..  1910  (5),  •^S(). 

«»Comp.  Rend.  Soc.  Biol.,  1910  (79),  OaS. 


INSECT  POISONS  15:^ 

that  spiders  without  liciiiolNsius  poison  flics  oxactly  as  those  with 
hemolysins. 

Von  Fiirth  considers  that  the  bite  of  the  historically  famous  Italian 
tarantula  is  able  to  cause  no  more  than  local  inflammation,  and  Ko- 
l)ert  found  that  the  entire  extract  of  six  Russian  tarantulas  (which 
are  supposed  to  be  more  poisonous  than  the  Italian)  caused  no  symp- 
toms when  injected  into  a  cat.  An  antitoxin  is  said  to  have  been 
secured  against  the  Russian  tarantula.^-'" 

In  all  probal)ility  the  other  poisonous  spiders  possess  toxic  sub- 
stances allied  to  those  of  the  venoms,  with  hemolytic,  agglutinative, 
and  neurotoxic  products,  Sachs'  studies  indicating  the  general  sim- 
ilarity of  all  tlie  zootoxins. 

Centipedes 

Undoubtedh'  the  severity  of  centipede  poisoning  has  been  greatly 
exaggerated,  the  results  being  usually  limited  to  local  inflammation, 
frequently  spreading  some  distance  in  an  erysipelas-like  manner. 
An  authentic  case  of  fatal  poisoning  of  a  child  four  years  old  by  a 
centipede  (Scolopendra  heros)  has  been  reported  from  Texas  by  G. 
Linceicum,*^  death  resulting  five  to  six  hours  after  the  bite  was  re- 
ceived. Besides  the  local  pain  and  inflammation,  vomiting  w^as 
marked,  occurring  also  in  five  other  non-fatal  cases. 

Centipedes  secrete  their  poison  in  relatively  large  glands,  which 
discharge  at  the  apices  of  a  pair  of  specialized  claws  that  take  the 
place  of  the  first  pair  of  legs.  The  nature  of  this  poison  seems  not 
to  have  been  investigated.  Numerous  chemical  substances  are  de- 
scribed as  secreted  bj'  other  glands  of  these  animals,  including  prus- 
sic  acid  and  a  camphor-like  matter  (see  v.  Fiirth). 

Bee  Poison 

Bee  poison  has  been  better  studied  than  mcst  insect  poisons,  begin- 
ning with  the  work  of  Paul  Bert  (1865).  It  is  secreted  by  the  glands 
into  a  small  poison  sac,  and  stored  up  until  ejected.  Cloez  found 
that  bee  poison  was  precipitated  by  ammonia,  tannin,  and  platinic 
chloride,  and  Langer  proved  it  to  be  a  non-volatile  organic  base.  As 
excreted,  it  is  acid,  contains  30  per  cent,  of  solids,  and  one  honey-bee 
secretes  0.0003-0.0004  gm.  It  contains  formic  acid  and  much  pro- 
tein, but  it  has  been  stated  that  the  poison  is  protein-free,  and  is  not 
destroyed  by  heat  (100°),  weak  acids,  or  alkalies.  On  the  other  hand, 
it  is  said  to  be  destroj-ed  by  proteolytic  enz3'mes,  which  would  indicate 
that  it  is  of  protein  nature.  Arthus**-  believes  the  evidence  indicates 
that  the  bee  venom  is  a  proteotoxin.  How^ever,  there  are  many 
points  of  resemblance  between  the  effects  of  insect  stings  and  the  local 

«"«  Konstanzoff,  Russky  Wratsch.,  1907,  Xo.  17. 
"  .\mer.  Jour.  Med.  Sci.,  1866  (52),  575. 
82  Jour.  Pharm.  Chim.,  1919  (20),  41. 


154  PHYTOTOXINS  AND  ZOOTOXINS 

effects  of  histamine  injection.*^  Hemolysis  is  produced  both  in  vitro 
and  in  vivo  with  all  sorts  of  blood,  but  to  very  different  degrees,  thus 
resembling  spider  toxin.  The  hemolytic  action  is  greatly  increased 
by  the  presence  of  lecithin,  forming  a  toxolecithid  like  "cobra  lecithid."^^ 
Locally  bee  poison  causes  necrosis,  with  marked  h3'-peremia  and  edema. 
A  4500  gm.  dog  was  killed  by  intravenous  injection  of  6  c.c.  of  a  1.5 
per  cent,  solution  of  pure  poison  (Langer).^^ 

Immunity  is  undoubtedly  possible,  for  bee-keepers  frequently  show 
a  great  decrease  in  susceptibility.  On  the  other  hand,  abnormally 
great  susceptibility  is  frequently  seen,  some  cases  of  fatal  poisoning 
having  been  observed. ^^  Dold***^"  was  unable  to  secure  experimental 
immunity  to  bee  poison. 

Wasps  and  Hornets  presumably  produce  poison-s  similar  to  those  of  the  bees. 
A  study  by  Bertarelli  and  Tedeschi*''  establishes  this  for  a  species  of  wasp  (Vespa 
crabro  L.). 

Ants  also  produce  formic  acid,  a  fact  so  well  known  that  it  has  come  to  be 
considered  that  this  is  the  source  of  their  toxicity.*^"  Von  Fiirth,  however,  sug- 
gests the  probability  that  ant  poison,  like  that  of  the  bees,  owes  its  chief  effects 
to  other  more  complex,  unknown  poisons.^* 

Lice. — Persons  bitten  by  large  numbers  of  lice  maj'-  exhibit  a  distinct  intoxica- 
tion, accompanied  by  an  eruption  resembling  measles.^*  The  nature  of  the  poison 
is  not  known,  but  it  does  not  produce  a  severe  local  urticaria  like  the  sting  of  bees 
and  wasps. 

Poisons  of  Dermal  Glands  of  Toads  and  Salamanders 

It  has  been  known  for  centuries  that  toads  produce  poisonous  sub- 
stances, Pare  in  1575  having  discoursed  interestingly,  if  inaccurately, 
on  this  topic.  Numerous  studies  have  been  made  of  these  poisons, 
which  are  secreted  by  the  dermal  glands  and  therefore  cannot  be  used 
for  poisoning  either  prey  or  enemies  (except  those  that  feed  upon 
them) ;  the  most  extensive  study  being  that  of  Faust. ^°  He  isolated 
two  constituents,  apparently  the  same  in  different  species  of  toads; 
one,  which  he  called  bufotalin,  is  very  active,  resembling  the  digitalis 
group;  the  other,  hufonin,  is  much  less  active.  Bufonin  is  neutral  in 
reaction,  soluble  in  warm  alcohol,  but  slightly  in  cold.     Analj^sis  in- 

83  See  Eppinger,  Wien.  klin.  Woch.,  1913  (26),  1413;  SoUmann  and  Pilcher, 
Jour.  Pharm.,  1917  (9),  309. 

8^  Morgenroth  and  Carpi,  Berl.  klin.  Woch.,  1906  (43),  1424. 

85  Arch.  exp.  Path.  u.  Pharm.,  1896  (38),  381;  Arch,  internat.  Pharniac.  et 
Ther.,  1899  (6),  181. 

8»  irospitaLstideude,  1905,  No.  27. 

8«"  Zeit.  Iiniiuinitiit.,  1917  (26),  284. 

8'  Cent.  f.  Jiakt.,  1913  (68),  309. 

8'"The  sting  of  nettles  is  said  to  be  due  to  formic  acid.      (See  Dobbin,  Xatur 
Sept.  18,  1919.) 

88  An  attempt  by  Barratt  (  Ann.  Trop.  Mi-d.  and  I'arasitol.,  1910  (4),  177)  to 
obtain  a  poison  from  culex  mosciuitos  was  unsuccessful.  The  l)odies  of  "black 
files"  contain  an  aiitive  poi.son  that  could  not  !>(>  ithMitified  by  Stokes  (Jour.  Cut. 
Di.s.,  191  I  (32),  830),  Ijcyond  thai  it  is  insolul)l('  in  alcohol,  which  does  not  in- 
activate it,  and  tiiat  it  is  destroyed  by  trypsin. 

8"  IJirschleldcr  and  Moore,  .Vrcli.  Int.'lMed.,  1919  (23),   119. 

»»  Arch.  f.  exp.  Path.  u.  Pharm.,  1902  (47),  279.  Complete  Ijihliugraphy  and 
review. 


POISONS  OF  TOADS  155 

dicates  an  empirical  I'oiiuuhi  of  C34H54O2.     It  probably  is  the  cause 
of  the  milky  appearance  of  the  dermal  secretion.     Bufotahn  seems  to 
be  C34H46O10,  is  acid  in  reaction,  soluble  in  chloroform  and  alcohol, 
but  not  in  petroleum  ether.     Subcutaneous  injection  of  2.G  mg.  bufo- 
talin  killed  a  dog  (weighing  4  kg.)  in  four  to  five  hours;  given  by 
mouth  it  causes  much  vomiting  and  diarrhea,  so  that  large  doses  are 
not  fatal.     It  causes  much  local  irritation  when  applied  to  mucous 
membranes,  but  produces  no  marked  change  at  the  site  of  injection. 
The  effects  on  the  circulation  resemble  in  all  respects  those  of  the  digi- 
talis group;  bufonin  acting  similarly  but  much  weaker  than  bufotalin. 
Bufotalin  seems  to  be  derived  from  bufonin  by  oxidation,  and  the  latter 
is  quite  similar  to  cholesterol,  apparently  having  the  following  formula: 
HO-H26C17-C17H26-OH.     An  important  consideration  is  that  Faust 
has  also  isolated  from  the  venom  of  cobra  and  crotalus,  poisons  which 
seem  related  to  these  toad  poisons,  the  cobra  poison  being  assigned  an 
empirical  formula  of  Ci7H260io,  and  the  crotalus  poison  C34H54O21. 
Fiihner^^  considers  bufotalin  to  be  more  closely  related  to  the  saponins. 
Phisalix  and  Betrand^-  have  found  poison  in  the  blood  of  toads 
similar  to  that  of  the  glands.     The  hemolytic  property  observed  by 
Pugliese^^  may  be  due  to  the  acidity  of  the  dermal  secretion.     The 
poisons  of  chfferent  species  seem  to  be  quite  the  same  in  all  (Faust). 
From  the  dermalsecretion  of  the  large  tropical  toad,  Bufo  agiia,  Abel 
and  Macht^*  have  isolated  two  distinct  active  substances;  one  identical 
with  epinephrine,  which  constitutes  nearly  seven  per  cent,  of  the  crude 
venom;  the  other,  which  makes  up  36  per  cent.,  is  called  hvfagin,  has 
a   composition  indicated  by  the  formula  C1SH24O0,  and  therefore  is 
presumably  related  to  the  rest  of  this  group  which  arises  from  choles- 
terol.    In  physiological  action  bufagin  resembles  digitalis,  and  it  is  ex- 
tremely active.     The  toad  is  relatively  immune  to  bufagin,  but  not 
at  all  to  the  epinephrine.     A  Chinese  drug  derived  from  toad  skins  has 
been   found  to  contain  similar  ingredients  (Shimizu^^),  as  well  as  a 
substance  resembling  picrotoxin  in  action. 

Salamanders  also  produce  poisonous  secretions  in  their  dermal  glands,  which 
have  been  studied  especially  by  Faust,^"  and  earlier  by  Zalesky,^^  who  isolated  an 
organic  base  which  he  named  samandarin.  Faust  describes  samandarin  as  first 
stimulating  and  then  paralyzing  the  automatic  centers  in  the  medulla.  The 
poison  resembles  the  alkaloids,  having  the  formula  C26H40N2O,  and  produces 
death  in  doses  of  0.7-0.9  mg.  per  kilo  (dogs)  with  respiratory  failure.  Immuniza- 
tion of  rabbits  was  practically  impossible.  A  second  alkaloid,  samandaridin 
(C-.uH.nXO)  is  also  present  in  even  greater  quantities  than  the  samandarin,  and 
differs  only  in  being  weaker. 

Frogs  also  have  similar  poisons  in  their  skins,  extracts  of  Rana  esculenta  skin 

91  Arch.  exp.  Path.  u.  Pharm.,  1910  (63),  374. 

9-  Arch.  d.  physiol.  norm,  et  path.,  1893  (5),  511. 

9'  Archivo  di  farm.  e.  terap.,  1894  (2),  321;  Arch,  ital  de  Biol.,  1895  (22),  79. 

"Jour.  Amer.  Med.  Assoc.  1911  (5lJ),  1531;  Jour,  of  Pharm.,  1912  (3),  319. 

95  Jour.  Pharmacol.,  1916  (8),  347. 

9«Arch.  e.xper.  Path.  u.  Pharm.  1898  (41),  229  (literature);  1900  (43),  84. 

9'  Hoppe-Seyler's  Med.  Chem.  Untersuch.,  1866,  p.  85. 


156  PHYrOTOXJX.s  AXD  ZOOTOXINS 

being  highly  toxic. '^*  The  dermal  secretions  of  most  of  the  an?phibians  are  poison- 
ous, not  only  for  mammals,  but  also  for  reptiles,  and  in  large  doses  for  the  animals 
producing  them  (Phisalixj."'-'  Bert'  and  also  Dutartre-  have  described  a  digitalis- 
like poison  in  the  secretion  of  the  dermal  glands  of  frogs. 

It  is  evident  that  all  these  poisons  are  quite  distinct  from  the 
venoms,  and  from  the  true  toxins,  apparenth'  being  simple  chemical 
compounds  not  related  to  the  proteins  and  not  capable  of  causing  im- 
munization. 

Poisonous  Fish  ^ 

There  are  numerous  fish,  especiall}'  in  tropical  waters,  which 
defend  themselves  by  injecting  poisons  into  their  enemies.  This  is  ac- 
complished by  spines,  to  which  are  attached  poison  glands.^  Dunbar- 
Brunton^  has  described  two  such  fish  {Trachinis  draco  and  Scorpoenn 
scorpha)  of  Mediterranean  waters.  Wounds  by  these  spines  cause  in 
animals  intense  local  irritation  and  edema  and  paralj'sis  of  the  part, 
followed  by  gangrene  about  the  site  of  the  wound;  in  fatal  poisoning 
death  occurs  in  from  one  to  sixteen  hours,  with  general  paralysis. 
The  sufferings  of  persons  so  poisoned  are  said  to  be  extreme,  and 
death  may  occur  either  directly  from  the  poison  or  later  from  sci:)sis 
following  the  local  gangrene.  Presumabl}'  this  poison  is  not  dissimilar 
to  that  of  the  snakes;  it  probably  is  not  an  alkaloid,  as  Dunbar- 
Brunton  suggests.  It  affects  chiefly  the  heart,  according  to  Pohl," 
and  contains  a  hemolytic  principle  which  behaves  like  the  venom 
hemolysins  in  that  it  is  activated  by  serum  (Evan)." 

Several  other  fish  secrete  poison  in  glands  attached  to  long  spines, 
one  of  the  most  poisonous  being  Synanceia  brachio,  which  is  known  to 
have  caused  fatal  intoxication  in  several  instances.  Only  the  Murw- 
nidce  seem  capable  of  poisoning  by  biting;  they  have  a  well-developed 
poison  apparatus  on  the  gums,  but  nothing  is  known  concerning  the 
poisons  they  produce. 

Many  fish  develop  poisonous  decomposition  products  remarkably 
soon  after  death,  especially  in  tropical  climates,  so  that  a  fish  that  is 
perfectly  wholesome  if  eaten  immediately  after  being  caught  may 
be  very  poisonous  if  kept  but  a  few  hours.  There  is  a  decided  differ- 
ence in  fish  of  different  varieties  in  this  respect,  so  that  some  cannot  be 
safely  marketed.  Some  of  the  poisonous  products  of  the  decomposition 
of  fish  seem  to  be,  early  products  of  protein  cU^avage,  of  liigli  inoh'cular 

»»  Caspari  and  Loewv,  Med.  Klinik,  19II  (7),  1204. 
"9  Jour.  Phvs.  et  Path,  gen.,  1910  (12),  325. 

'  Compt.  Rend.  Soc.  Biol.,  1885,  p.  524. 

2  Ibid.,  1890,  p.  199. 

^  Full  discu.ssion  and  literature  given  by  Faust,  "Tierische  Giftc,"  p.  134. 

*  For  a  list  of  fLsh  with  poison  glands  see  Pawlowskv,  Zool.  Jahrb.,  1912  (31), 
529. 

»  Lancet,  189(i  (ii),  (iOO. 

•  Prager  med.  Woch.,  1893  (18),  31. 
'  British  Me<l.  Jour.  1907  (i),  73. 


POISONOUS  FISH  157 

complexity,  I'oi-  llicy  aic  dificsfcd  by  pepsin  and  trypsin,  but,  not  by 
erepsin.** 

There  are  also  other  tisli  wliose  botUes,  even  wlien  perfectly  fresh, 
contain  very  powerful  poisons.  Savtschenko,^  in  his  elaborate  atlas 
of  th(!  poisonous  fish  describes  a  nunibei-  of  cases  of  poisoning  by  the 
famous  "parrot  fish"  of  Japan  (Tc/rof/ow),  in  which  the  poison  seems 
to  be  developed  and  contained  in  the  ovaries  and  eggs,  and  therefore 
the  degree  of  toxicity  varies  with  the  season  of  the  year  in  which  the 
fish  is  taken.'"  Poisoning  by  these  fish  is  very  violent,  the  symptoms 
appearing  quickly,  and  the  cases  are  divided  into  two  groups  b\'  Savt- 
scheidvo.  as  the  algid,  or  choleriform,  and  the  gastro-intestinal  type. 
The  symptoms  of  the  algid  form  appear  almost  immediately  after 
eating  the  fish,  and  consist  of  pain  in  the  stomach,  with  great  fear  and 
distress;  soon  diarrhea  and  vomiting  set  in,  with  cramps  in  the  arms 
and  legs;  this  terminates  in  collapse,  coma,  and  death  from  either  re- 
spiratory or  cardiac  paralysis.  The  entire  course  of  the  process  may 
be  but  ten  to  twenty  minutes,  or  it  may  be  as  many  hours.  On 
account  of  the  localization  of  the  poison  in  the  eggs  and  ovaries  not 
all  persons  who  eat  the  fish  are  poisoned,  and  not  all  who  are  poisoned 
receive  a  fatal  dose.  In  the  gastro-intestinal  form  the  symptoms  appear 
later,  consist  chiefly  of  gastro-intestinal  disturbances  resembling  more 
closely  ptomain  poisoning,  and  the  prognosis  is  not  so  bad  as  in  the 
algid  form. 

The  pathological  anatomj^  of  this  form  of  poisoning  has  not  been 
carefully  studied,  but  no  characteristic  or  striking  anatomical  changes 
have  been  noted  in  the  bodies  examined.  Tahara"  has  described  a 
toxic  body,  tetrodo-toxin,  isolated  from  the  ovaries  of  Tetrodon.'^^ 
The  purest  preparations  had  a  minimum  lethal  dose  of  0.0025  to  0.004 
gm.  per  kilo,  and  a  provisional  formula  of  CieHaiNOie  was  given  to  it. 
Tetrodotoxin  is  neither  protein  nor  alkaloid,  nor  yet  a  protamin. 
It  anesthetizes  motor  nerve  endings  and  central  nervous  system, 
paralyzes  both  motor  and  sensory  nerves,  increases  the  excitability  of 
muscle  (Itakura)'^  and  paralyzes  sympathetic  nerve  endings  (Ishihara). 

In  this  connection  may  be  mentioned  the  peculiar  erysipelas-like 
lesions  caused  by  bites  of  crabs,  which  indicates  the  formation  of  some 
toxic  product  by  these  crustaceans.'*  Gilchrist'^  obtained  a  history 
of  l)ites  or  injuries  by  crabs  in  323  of  329  cases  of  "erysipeloid." 

s  Konstanzoff  and  Manoiloff,  Wien.  klin.  Woch.,  1914  (27),  883. 

*  "Atlas  des  Poissons  Veneneu.x,"  St.  Petersburg,  1886  (literature). 

^^  A  Brazilian  fish,  Spheroides  testudineus,  has  extremely  toxic  tissues,  extract 
of  0.01-0.02  gm.  of  liver  killing  a  guinea  pig  in  a  few  minutes  (Fonseca,  Brazil- 
Medico,  1917  (31),  97).  The  ovaries  of  the  American  gar  are  also  said  to  be  toxic 
(Greene  et  al,  Amer.  Jour.  Phv.s.,  1918  (45),  558). 

'1  Biochem.  Zeit.,  1910  (30),  2.56. 

'2  Arch.  exp.  Path.  u.  Pharm.,  1890  (26),  401  and  453. 

'3  Mitt.  med.  P\ak.  Univ.  Tokio,  1917  (17),  455. 

'^  The  livers  of  the  spiny  lobster  (genus  Pouvus)  have  been  found  to  be  very 
toxic  (Nakano,  Jap.  Ztschr!  Dermatol.,  1917  (17),  1). 

'^  Jour.  Cutaneous  Diseases,  November,  1904. 


158  PHYTOTOXINS  AND  ZOOTOXINS 

Crabs,  in  turn,  may  be  poisoned  by  cephalopods  which  secrete  an  active 
poison  from  their  sahvary  glands.'^  INIany  coelenterates  produce 
active  poisons  (most  famous  of  these  being  the  Portuguese-man-o'-war) , 
which  have  especially  a  paralyzing  and  a  local  irritant  effect. ^^ 

Eel  Serum 

In  1888  Mosso'^  studied  the  toxic  properties  of  eel  serum,  which  he  found  was 
extremely  poisonous  for  experimental  animals,  0.1  to  0.3  c.c.  per  kilo  being  fatal 
for  rabbits  and  dogs  in  a  few  minutes  if  intravenously  injected;  introduced  into 
the  stomach  it  is  not  toxic,  but  it  produces  a  violent  conjunctivitis  when  it  enters 
the  eye,  the  poisonous  agent  being  contained  in  the  albumin  fraction.''  The 
poisonous  principle  Mosso  called  ichthyotoxin.  Death  results  from  respiratory 
failure  with  large  doses;  small  doses  lead  to  cachexia  and  death  after  a  few  days. 
The  coagulability  of  the  blood  is  greatly  reduced.  Kossel-"  found  histological 
changes  in  the  central  nervous  system  in  such  animals,  that  resembled  the  lesions 
of  tetanus.  He  succeeded  in  securing  an  active  antitoxin  which  neutralized  the 
strongly  hemolytic  action  of  eel  serum  in  vitro,  and  also  prevented  fatal  effects 
in  animals.  Camus  and  Gley-'  have  studied  the  physiological  action  of  eel  serum 
and  found  it  strongly  hemolytic,  and  also  apparently  neuro-toxic.  The  toxicity 
is  destroyed  by  heating  to  58°  for  fifteen  minutes.  By  immunization  an  antitoxic 
serum  can  be  obtained  which  neutralizes  the  eel  toxin  completelj\  Tchistovitch-- 
secured  antitoxic  seram,  which  acted  also  as  a  precipitin  for  eel  serum.  De  Lisle^^ 
found  that  eel  serum  does  not  act  like  an  amboceptor,  since  after  heating  it  cannot 
be  reactivated  with  fresh  mammalian  serum,  and  it  seems,  therefore,  to  be  different 
from  snake  serum  in  its  structure. ^^  Lamprey  serum  is  likewise  toxic,'*  as  is 
also  that  of  the  Rays.  Not  only  the  serum,  but  also  the  palate  glands  of  the  Moray 
(Murcena  helena)  contain  toxic  antigenic  substances  resembling  snake  venoms 
(Kopacz  e  wski) .  ^^ 

I''  Baglioni,  Zeit.  f.  Biol.,  1908  (52),  130. 

1''  See  von  Fiirth,  Vergl.  chem.  Phvsiol. ;  also  Lojacono,  Jour.  d.  physiol.,  1908 
(10),  1001. 

18  Arch.  Ital.  de  Biol.,  1888  (10),  141;  1889  (12),  229. 
"Pollot  and  Rahlson,  Graefe's  Arch.,  1911  (72),  183. 

20  Berl.  klin.  Woch.,  1898  (35),  152. 

21  Arch,  internat.  d.  Pharm.,  1899  (5),  247. 

22  Ann.  Inst.  Pasteur,  1899  (13),  406. 

23  Jour,  of  Med.  Research,  1902  (8),  396. 

2^  Corroborated  by  Sato,  Nippon  Biseibutsugakkai  Zassi,  1917  (5),  No.  35. 
25  Gley,  Compt.  Rend.  Soc.  Biol.,  1915  (78),  116;  Camus  and  Gley,  ibid.,  p.  203. 
2«  Compt.  Rend.  Acad.  Sci.,  1917  (165),  513;  Ann.  Inst.  Pasteur,  1918  (32),  584. 


CHAPTER  VII 

CHEMISTRY    OF    THE    IMMUNITY    REACTIONS— ANTIGENS, 
SPECIFICITY,    ANTITOXINS,   AGGLUTININS,   PRECIPI- 
TINS,  OPSONINS,   AND   RELATED   SUBJECTS 

Although  immunitj^  was  first  investigated  in  relation  to  bacterial 
infection,  it  was  soon  learned  that  the  reactions  by  which  the  animal 
body  defends  itself  against  bacteria  have  not  been  developed  as 
specific  means  of  defense  against  bacteria  alone,  but  are  reactions 
against  foreign  substances  of  similar  chemical  nature,  whether  bac- 
terial, animal,  vegetable  or  artificially  synthetic  in  origin.  Further- 
more, these  reactions  are  chemical  reactions,  and  the  problems  of 
immunity  are  chemical  problems,  although  as  yet  most  of  the  react- 
ing substances  are  not  accessible  to  chemical  investigation.  In  this 
place,  where  our  concern  is  with  the  chemical  aspects  of  pathological 
processes,  the  subject  of  immunity  will  be  discussed  only  from  the 
standpoint  of  the  chemistry  of  the  processes  and  substances  involved, 
leaving  to  other  works  the  clinical  and  bacteriological  aspects  of  the 
subject.' 

The  reactions  of  immunity  are,  we  find,  reactions  to  chemical  sub- 
stances entering  the  body  from  without,  or  abnormally  developed 
within  the  body  by  invading  organisms  or  by  changes  in  the  chemi- 
cal processes  of  the  body.  Furthermore,  there  seems  to  be  an  essential 
difference  between  the  reactions  incited  by  simple  chemical  compounds 
to  which  the  animal  body  can  develop  a  certain  degree  of  resistance 
(such  as  morphine,  alcohol,  and  arsenic),  and  the  reactions  against 
more  complex  substances  such  as  bacterial  toxins,  foreign  proteins, 
venoms,  etc.  The  complex  substances  of  the  latter  group  incite 
reactions  which  are  to  a  greater  or  less  degree  specific,  and  usually 
very  highly  augment  the  defense  of  the  body  against  the  foreign  sub- 
stances; with  the  simple  poisons  the  reactions  are  largely  or  altogether 
non-specific,  and  the  resulting  resistance  is  relatively  slight.  Sub- 
stances of  the  first  class  we  refer  to  as  antigens. 

'  Especially  to  be  recommended  for  a  discussion  of  the  scientific  problems  of 
immunology  is  Zinsser's  "Infection  and  Resistance,"  Macmillan,  New  York,  1914; 
and  for  methods  and  applications  see  Kolmer's  "Infection,  Immunity  and  Specific 
Therapy,"  W.  B.  Saunders,  Philadelphia,  1918.  Also  see  Kolle  and  Wassermann, 
"Handbuch  der  path.  Mikroorganismen;"  Weichardt  "  Jahresbericht  der  Immuni- 
tatsforschung." 

159 


160  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

ANTIGENS- 

This  term  includes  those  substances  which,  when  introduced  into 
the  blood  or  tissues  of  an  animal,  in  proper  amounts  and  under  suitable 
conditions,  cause  the  generation  and  appearance  in  the  blood  of 
specific  antibodies  capable  of  reacting  with  the  antigen.^  Concerning 
the  chemistry  of  antigens  we  can  say  that  all  antigens,  so  far  as  now 
known,  are  colloids.  Furthermore,  with  one  exception,  every  known 
soluble,  complete  protein  may  act  at  least  to  some  degree  as  an  anti- 
gen, and,  as  yet,  it  has  not  been  finally  established  that  any  colloids 
other  than  proteins  can  act  as  antigens.  The  exception  is  the  race- 
mized  protein  of  Dakin,  which  Ten  Broeck^  found  to  be  entirely 
non-antigenic  although  soluble  and  possessed  of  all  the  amino-acids 
present  in  the  egg  albumin  used  in  preparing  it.  Solubility  is  an  es- 
sential character  for  antigenic  action,  for  proteins  that  have  been  coagu- 
lated by  heat  lose  their  antigenic  capacity,  while  proteins  that  are  not 
coagulated  (e.  gf.,  casein,  ovomucoid)  retain  their  antigenic  properties 
after  boiling.^  A  tj^pical  incomplete  protein,  gelatin,  is  devoid  of 
antigenic  power  (Starin).^  The  same  is  true  of  the  protamines  and 
histones.''     Hemocyanin,  however,  is  antigenic  (Schmidt)."" 

Of  the  cleavage  products  of  proteins  it  is  certain  that  none  of  the 
amino-acids  and  simple  polypeptids  can  act  as  antigens,  and  even  such 
large  complexes  as  the  proteoses  are  antigenic  to  but  a  slight  degree  if 
at  all.*  Whether  the  entire  protein  molecule,  or  onh'  groups  thereof, 
determine  the  characteristics  of  the  antigen,  is  not  known,  there  being 
evidence  which  can  be  interpreted  in  favor  of  either  view,  but  Wells  and 
Osborne^  have  submitted  evidence  which  indicates  that  a  single  pure 
protein  can  act  with  and  engender  more  than  one  antibody;  this  is 
supported  by  Klein's  demonstration  of  the  production  of  two  distinct 
antibodies  by  immunizing  with  casein.'''    * 

There  seems  to  be  no  very  definite  relation  between  the  amount  of 
antigen  injected  and  the  amount  of  antibody  produced."  Neverthe- 
less Herzfeld  and  Klinger'-  have  advanced  the  hypothesis  that  the 
antibodies  are  really  fragments  of  the  antigen  molecules  which  have 

^  See  the  Review  on  Antigens  by  E.  P.  Pick,  Kolle  and  Wassermann's  Handbuch 
d.  path.  Mikroorganismen,  1912  (1),  G85. 

'  Attempts  to  influence  the  capacity  to  produce  antibodies  by  modifying  diet 
have  not  produced  striking  results.     (See  Zilva,  Biochem.  Jour..  1919  (13),  172.) 

*  Jour.  Biol.  Chem.,  1914  (17),  369;  also  Schmidt,  Proc.  Soc.  Exp.  Biol.  Med., 
1917  (14),  104;  Kahn  and  McNeil." 

^  See  Wells,  Jour.  Infect.  Dis.,  1908  (o),  449;  Jour.  Biol.  Chem.,  1916  (28),  11. 

"  Jour.  Infect.  Dis.,  1918  (23),  139;  corroborated  by  Kahn  and  McNeil,  Jour. 
Immunol.,  1918  (3),  277. 

'  Wells,  Zeit.  Immunitiit.,  1913  (19),  599;  Schmidt,  Jour.  Infect.  Dis.,  1919 
(2.5),  207. 

'"Proc.  Soc.  Biol.  Chem.,  1919  (14)  Ixix;  Jour.  Biol.  Chem.,  1920  (41). 

'  See  Fink,  ,Jour.  Infect.  Dis.,  1919  (25),  97;  full  review  on  proteoses  as  antigens. 

9  Jour.  Infec.  Dis.,  1913  (12),  341. 

'»  Folia  Microbiol.    1912  (1),  101. 

"  See  Tsen,  .Jour.  Med.  lies.,  1918  (37),  381. 

'2  Biochem.  Zeit,  1918  (85),  1. 


NON-PROTEIN  ANTIGENS  161 

been  absorbed  by  the  blood  proteins,  whereas  Liebermann'^  suggests 
that  they  are  altered,  liquefied  portions  of  the  cells  which  the  antigens 
have  attacked. 

It  has  been  shown  by  Gay  and  Robertson'*  that  if  the  non-anti- 
genic  cleavage  products  of  casein  are  resynthesized  by  the  reverse 
action  of  pepsin,  into  a  protein  resembling  paranuclein,  this  synthetic 
protein  is  capable  of  acting  as  an  antigen.  Protamins  and  globin,  they 
found,  were  not  antigenic,'"  although  globin  when  combined  with  casein 
forms  a  compound  which  engenders  an  antibody  that  gives  complement 
fixation  reactions  with  globin.  Schmidt  also  found  that  protamin 
edestinatc  is  antigenic  for  edestin  and  for  itself,  but  not  for  protamins, 
whereas  a  compound  protein,  both  elements  of  which  were  non-anti- 
genic  (globin-albumose),  was  not  antigenic."' 

NON-PROTEIN  ANTIGENS 

Among  the  many  accounts  of  what  the  authors  interpret  as  the 
successful  production  of  specific  antibodies  as  a  reaction  to  non-pro- 
tein antigens,  are  the  following: 

Ford'"  found  that  rabbits  can  be  immunized  to  extracts  of  Aman- 
ita phaUoides,  and  that  the  serum  of  such  rabbits  will  neutralize  five 
to  eight  times  the  lethal  dose  for  guinea-pigs,  and  is  anti-hemolytic 
for  the  hemolysin  of  amanita  when  diluted  to  1-1000.  As  he  and 
Abel"-^  had  found  this  hemolytic  poison  of  Amanita  to  be  a  glucoside, 
this  observation  is  to  be  interpreted  as  a  successful  production  of  an 
antibod}^  for  a  non-protein  poison,  a  glucoside.  This  work  was  fur- 
ther supported  by  successfully  immunizing  rabbits  to  extracts  of 
Rhus  toxicodendron,  and  finding  that  their  serum  in  doses  of  1  cc. 
will  protect  guinea-pigs  from  5-6  lethal  doses  of  the  poison,  which 
was  found  by  Acree  and  Sjane^^  to  be  a  glucoside.  Subsequent  work 
by  the  same  author  confirms  the  main  point,  showing  that  an  active 
hemolysin  can  be  obtained  free  from  demonstrable  protein,  and  that 
immunization  with  this  protein-free  hemolysin  will  result  in  strongly 
active  (1-1000)  antihemolytic  serum.^o  The  antihemolysin  unites 
with  the  hemolysin  in  simple  multiple  proportions. ^^  Another,  non- 
hemolytic poison  from  Amanita,  which  Ford  designates  as  Amanita 
toxin,  was  found  to  contain  neither  protein  nor  glucoside,  and  no 
antitoxic  serum  or  definite  artificial  immunit}'  can  be  obtained  for  it. 

Jacoby  believed  that  he  had  obtained  the  phytotoxin  ricin  free  from 

"/Wd.,  1918  (91),  46. 
;7  1^  Jour.  Biol.  Chem.,  1912  (12),  233. 

IS  Jour.  Exp.  Med.,  1912  (16),  479;  1913    17),  535. 

"  Univ.  of  Calif.  Publ.,  Pathol.,  1916  (2),  157.  Review  and  bibliography  on 
specificity. 

"  Jour.  Infec.  Dis.,  1907  (4),  541. 
"  Jour.  Biol.  Chem.,  1907  (2),  273. 

19  Jour.  Biol.  Chem.,  1907  (2),  547. 

20  Jour.  Pharmacol.,  1910  (2),  145. 
"  Jour.  Pharmacol.,  1913  (4),  235. 

11 


162  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

protein,  in  which  case  the  well-known  and  active  antiricin  must  rep- 
resent an  antibody  for  a  non-protein  antigen.  However,  the  work 
of  Osborne,  Mendel  and  Harris22  has  shown  that  ricin  is,  in  all  proba- 
bility, an  albumin,  and  this,  for  the  present  at  least,  places  ricin  with 
the  protein  antigens.  Nucleic  acid  and  nucleinates  have  been  found 
to  be  non-antigenic  ( Wells, ^^  Taylor). 

The  work  of  Ford  is,  in  our  estimation,  the  strongest  evidence  yet 
presented  as  to  the  possibihty  of  non-protein  antigens.  The  newer 
developments  in  immunological  research,  moreover,  make  it  seem 
entirely  plausible  that  a  complex  glucoside,  which  can  be  hydrolyzed 
by  enzymes,  can  act  as  an  antigen.  If  we  consider  the  evidence  that 
immunity  consists  in  the  development  of  a  special  power  to  hydro- 
lyze  foreign  substances,  when  these  substances  are  of  such  a  nature 
as  to  stimulate  the  cells  to  activity,  and  that  Abderhalden  and  others 
have  found  evidence  that  specific  enzymatic  properties  appear  in 
the  blood  of  animals  injected  with  carbohydrates  and  fats,  it  seems 
entirely  reasonable  that  a  toxic  glucoside  can  have  antigenic  proper- 
ties. A  similar  line  of  reasoning  will  apply  to  the  question  of  lipoid 
antigens. 

Lipoids  as  Antigens. — The  evident  participation  of  lipoids-^  in  immunity  reac- 
tions, especially  the  complement-fixation  and  allied  reactions,  has  naturallj''  led 
to  investigation  of  the  possibility  that  lipoids  may  act  as  true  antigens,  a  possi- 
bility made  conspicuous  by  the  fact  that  lipoids  can  be  substituted  for  true  antigens 
in  the  Wassermann  reaction  {q.  v.).  Bang  and  Forssmann  immunized  with  ethereal 
extracts  of  red  corpuscles  and  obtained  hemolysins,  so  that  they  concluded  that 
the  antigenic  constituent  of  the  corpuscles  is  a  lipoid,  probably  a  phosphatid. 
This  work  has  caused  much  controversy  and  manj^  workers  have  failed  to  confirm 
their  results. ^^  It  is  a  striking  fact  that  when  purified  phosphatids,  from  sources 
favorable  for  obtaining  pure  materials,  are  used,  the  results  are  usually  negative, 
while  the  positive  results  are  generally  reported  with  lipoids  of  more  or  less  dubious 
purity. 

"Nastin,"  the  lipoid  material  from  a  streptothrix,  has  been  used  by  Much  and 
others,  who  state  that  sera  are  obtained  which  give  complement  fixation  reactions 
with  nastin  used  as  the  antigen. ^^  Similar  results  are  described  for  the  fatty 
materials  from  tubercle  bacilli  ("tuberculonastin").  Warden^'  reports  securing 
positive  precipitin  and  fixation  reactions,  not  only  with  fatty  complexes  from 
bacteria  and  red  cells,  but  also  with  artificial  nii.xturcs  of  soaps  made  up  to  resemble 
the  cellular  lipins;  indeed,  he  states  that  the  lipoidal  antigens  are  more  specific 
than  proteins,  and  infers  that  the  specificity  of  antibodies  is  in  part  or  wholly  due 
to  the  fats  of  the  cells. 

Meyers^*  has  reported  the  production  of  specific  complement  fixation  antibodies 
by  immunizing  rabbits  with  acetone-insoluble  lipoidal  material  obtained  from  tape 
worms  and  cchinococcus.  He  has  found  the  acetone-insoluble  fraction  of  tubercle 
bacilli,  presumably  phosphatids,  to  serve  as  antigen  in  complement  fixation  reactions 

"  Amer.  Jour.  Physiol.,  1905  (14),  259. 

"Zeit  Immunitut.,  1913  (19),  599.  Lichtenstein  (Arch.  Physiol.,  1915,  p. 
189)  claims  to  have  produced  agglutinins  for  spermatozoa  and  yeasts  with  sodium 
nucleinate  from  sixtim  and  yeasts. 

^*  Bibliography  on  LijK)ids  and  Immunity  given  by  Landsteiner,  KoUe  and  Was- 
serniann's  llaiRll)uch,   19i;}  (2),    1210;  Jobling.  Jour.  Immunol.,   191G  (1),  491. 

'^  Review  of  literature  by  I.andsteiner,  Jahresb.  ImmunitJitsfrsch.,  1910  (6), 
209.     See  also  Hemolysis,  (!hap.  ix. 

"  Literature  in  Jioitr.  Klinik  d.  Tiiberk.,  1911  (20),  341. 

"Jour.  Infect.  Dis.,  19I.S  (22),  133;  (23),  501;  1919  (24),  285. 

"Zeit.  IiumunitiLt.,  1910  (7),  732;  1911  (9),  530;  1912  (14), 355. 


LIPOIDS  AS  ANTIGENS  163 

with  antibodies  for  tubercle  bacilli,^*  and  much  more  effectively  than  the  protein 
residue  of  the  bacilli,  wherefore  he  concludes  that  the  reactions  obtained  with 
the  lipoids  certainly  cannot  be  ascribed  to  adherent  traces  of  protein.  BergeP" 
observed  after  lecithin  injections  in  rabbits,  not  only  an  increase  in  the  lipase  con- 
tent of  the  blood  and  tissues,  but  also  the  presence  of  complement-binding  anti- 
bodies, and  Jobling  and  Bull"  have  found  an  increase  in  serum  lipase  after 
immunizing  with  red  corpuscles.'* 

The  number  of  reputed  positive  results  with  lipoids  makes  it  impossible  at  this 
time  to  state  dogmatically  that  lipoids  may  not  possess  antigenic  properties,  but 
it  must  be  taken  into  account  that  the  successful  use  of  lipoids  as  antigens  in  comple- 
ment fi.xation  reactions  is  not  proof  of  their  true  antigenic  nature,  in  view  of  our 
present  lack  of  knowledge  of  the  actual  nature  of  this  reaction  itself.  MacLean,'' 
indeed,  found  evidence  that  even  in  the  Wa-ssermann  reaction  the  active  substance 
is  not  lecithin  itself,  but  some  other  unknown  substance  which  could  be  obtained 
practically  lecithin-free.  Furthermore,  we  have  the  testimony  of  Fitzgerald 
and  Leathes^'*  that  a  lipoidal  material  from  liver,  which  was  itself  capable  of  serving 
as  antigen  in  the  Wassermann  reaction,  did  not  engender  complement-fixing 
antibodies  in  rabbits  immunized  with  this  lipoid.  Ritchie  and  Miller"  could  find 
no  antigenic  activity  in  the  lipoids  of  serum  or  corpuscles.  Also  Kleinschmidt,'® 
who  accepts  the  antigenic  nature  of  nastin,  was  unable  to  secure  antibodies  by 
immunizing  rabbits  with  it.  Thiele"  says  that  as  lipoids  possess  no  specificity 
they  cannot  give  rise  to  antibodies.  Neufeld  found  that  rabbits  immunized  with 
lecithin  developed  no  opsonins  for  lecithin  emulsions.  A  suggestive  observation  is 
that  of  Pick  and  Schwarz,'*  who  found  that  the  presence  of  lecithin  increases  the 
antigenic  power  of  bacteria,  which  may  help  to  explain  the  activity  of  possible  traces 
of  proteins  in  lipoid  preparations  used  as  antigens. 

Simple  Chemical  Antigens. — Many  drugs  cause  a  hypersensitization,  and  in 
this  respect  seem  to  behave  as  antigens  producing  anaphylactic  antibodies.  It 
happens  that  most  of  these  chemicals  are  of  such  a  nature  as  to  permit  of  their 
union  with  proteins,  and  it  seems  probable  that  such  protein  compounds  behave 
as  foreign  proteins  to  the  animal  in  which  they  are  formed,  for  it  has  been  found 
that  guinea-pig  serum  treated  with  iodin  can  render  guinea-pigs  sensitive  to  the 
same  iodized  serum. '^  Hence,  hypersensitiveness  to  iodin  compounds  would  be  a 
reaction  to  iodized  proteins,'*"  and  not  to  the  non-protein  iodin  compound;  the 
same  applies  to  anaphylactic  reactions  observed  with  salvarsan,  atoxyl.^i  copper 
compounds,**  and  perhaps  aspirin  and  antipyrin.*'  Zieler,  however,  has  ques- 
tioned the  validity  of  many  of  the  experiments  on  which  these  views  are  based.  ■•* 
It  is  possible  that  certain  chemicals  may  react  in  such  a  way  with  the  tissue  or  blood 
proteins  as  to  make  them  sensitive  to  the  animal's  own  complement,  which  then 

2^J*w^a912  (14),  359;  1912  (15),  245. 

30  mxit.  .\rch.  klin.  Med.,  1912  (106),  47. 

'1  Jour.  Exp.  Med.,  1912  (16),  488. 

'*  Bogomolez  suggests  that  the  lipoids  themselves  may  be  produced  in  excess 
for  defense  against  various  poisons,  which  they  serve  to  inhibit,  especiallj-  the 
toxin  of /i.  hotulinus.  (Zeit.  Immunitat.,  1910  (8),  35). 

'*  Lecithin  and  Allied  Substances,  Biochemical  Monographs,  1918,  p.  170. 

^^  Univ.  of  Calif.  Publ.,  1912  (2),  39. 

"  Jour.  Path,  and  Bact.,  1913  (17),  429;  but  Wang.  {Ibid.,  1919  (22),  224) 
obtained  positive  results  with  ether-chloroform  extracts  of  corpuscles. 

'«  Berl.  klin.  Woch.,  1910  (47),  57. 

"Zeit.  Immunitat.,  1913  (16),  160. 

38  Biochem.  Zeit.,  1909  (15),  453. 

39  Friedberger  and  Ito,  Zeit.  Immunitat.,  1912  (12),  241. 

*"  According  to  Block  (Zeit.  exp.  Path.,  1911  (9),  509)  iodoform  idiosyncrasy 
depends  upon  the  CH3  rather  than  on  the  iodin,  and  is  a  local  cellular  reaction 
rather  than  a  humoral  reaction,  the  protoplasm  having  an  increased  affinity  for 
methyl  radicals.     (See  Weil,  Zeit.  Chemotherapie,  1913  (1),  412.) 

*^  Moro  and  Stheeman,  Miinch.  med.  Woch.,  1909  (56),  1414. 

^-  Hollande,  Compt.  Kend.  Soc.  Biol.,  1918  (81),  58. 

"  Bruck,  Berl.  klin.  Woch.,  1910  (47),  1928;  Klausner,  Munch,  med.  Woch., 
1911  (58),  138. 

"  Miinch.  med.  Woch.,  1912  (59),  401. 


164  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

forms  anaphylatoxin,''"  and  thus  causes  reactions,  but  the  whole  anaphylatoxin 
question  is  in  so  uncertain  a  state  at  the  time  of  writing  that  further  sf>eculation  in 
this  direction  is  not  justifiable. 

The  attempts  to  produce  antitoxin  against  cantharidin  have  not  yielded  con- 
vincing results/"  nor  against  einnephrine.^'  De  Angelis^*  claimed  that  he  had 
produced  specific  precipitins  for  various  natural  and  sj'nthetic  dyes,  but  this  work 
has,  as  was  to  be  expected,  failed  of  confirmation.^^  Elschnig  and  Salus^"  state 
that  melanin  from  the  eye  is  antigenic,  producing  complement-fixing  antibodies 
specific  for  melanin  but  not  for  the  si)ecies.  Woods^'  has  corroborated  this  and 
also  demonstrated  anaphylactic  sensitization.  We  know  too  little  concerning  the 
composition  of  melanin  to  interpret  these  observations. 

'^  In  general  terms,  therefore,  antigens  ai'e  protein  molecules,  and 
the  reactions  of  immunity  are  reactions  against  proteins  foreign  to 
the  body  of  the  host,  and  manifested  by  the  presence  in  the  blood 
of  the  reacting  animal  of  substances  which  combine  with  and  cause 
recognizable  changes  in  the  foreign  protein.^-  These  changes  are 
recognized  in  many  ways,  such  as  precipitation,  agglutination,  com- 
plement-fixation, etc.,  and  the  question  at  once  arises  as  to  whether 
these  different  manifestations  depend  each  upon  a  separate  antibody, 
or  if  several  or  all  of  them  are  not  caused  by  a  single  antibody,  the 
action  of  which  is  indicated  by  the  different  reactions  which  are  made 
manifest  by  different  procedures  in  each  case.^^  This  question  will  be 
discussed  further  in  later  paragraphs. 

Knowing  that  the  antigens  are  merely  foreign  proteins  which  have 
been  introduced  into  the  body  of  an  animal,  there  naturalh'  occurs 
the  thought  that  the  animal  body  is  continually  receiving  in  its  food 
foreign  proteins,  and  against  which  it  defends  itself  in  the  alimentarj- 
canal  by  enzymatic  action,  which  disintegrates  these  proteins  until 
they  have  lost  their  colloidal  character.^-*  Logically  following  this 
comes  the  idea  that  perhaps  the  reactions  of  immunitj'  are  simply 
the  same  or  similar  disintegrative  enzymatic  actions,  carried  on  within 
the  blood  and  tissues  to  protect  the  body  in  the  same  way  against 
foreign  proteins  which  the  alimentary  digestive  apparatus  has  not 
had  the  opportunity  to  destroy.  This  conception  of  the  nature  of  im- 
mune reactions  to  antigens  has  been  especially  advanced  and  in- 

«  See  Manoilov,  Wicn.  klin.  Woch.,  1912  (25),  1701. 

«  Champy,  Compt.  Rend.  Soc.  Biol.,  1907  (G2),  1128. 

"  PoUak,  Zeit.  physiol.  Chem.,  1910  (GS),  C9. 

"  Ann.  di  Ig.  Spcrim.,  1909  (19),  33. 

"  Takemura,  Zeit.  Immunitiit.,  1910  (5),  697. 

*"  Graefe's  Arch.,  1911  (79),  428. 

"  Jour.  Immunol.,  1918  (3),  75. 

^^  Drew  lias  found  no  evidence  of  antibody  formation  l\v  immunizing  molluscs 
and  ecliinodcrms  (Jour,  of  Hyg.,  1911  (11),  188),  from  which  he  concludes  that 
the  reaction  to  foreign  jjroteins  is  not  a  universal  projierty  of  protoplasm;  a  sweep- 
ing generalization  which  requires  more  extensive  invest iagt ion  for  its  establish- 
ment. C'antacuzene  (C'oni])t.  Kend.  Soc.  Hiol.,  1913  (74),  111)  obtained  precipitins 
by  immunizing  J'/i(illusiii  nunnilhita  with  mammalian  l)K)i)d,  l)ut  no  hemolysins 
with  tiiis  or  AjthrodUe  (iculrald  and  Elcdone  inoschata. 

"See  Dean,  Lancet,  Jan.  13,  1917. 

"  Carrel  and  Ingebrigsten  (Jour.  Exp.  Med.,  1912  (15).  287)  have  found  that 
tissues  growing  in  vitro  witli  foreign  blood  produce  iiemolytic  antil>odies  for  that 
blood,  indicating  that  isolated  cells  can  react  to  antigens  l)v  anlilxuly  production. 


SPECIFICITY  1G5 

vcstifiiatecl  by  \'ic'tor  (\  \'auji;liair''  and  l)is  co-workers,  and  l)y  ImiiII 
Abdcrhalden,  who  has  demonstrated  in  various  ways  an  increased 
proteolytic  power  in  the  blood  of  animals  which  have  received  pa- 
renteral injections  of  foreign  proteins.^"  Thus,  if  the  antiserum  re- 
acts on  the  specific  proteins  within  a  dialyzin<!;  sac,  the  products  of 
proteolysis  diffuse  into  the  surrounding  medium  where  they  can 
be  detected  by  simple  chemical  reactions.  Also,  changes  in  the  spe- 
cific rotation  of  the  protein  or  peptid  solution  can  be  observed  by 
the  polariscopic  reading  before  and  after  the  action  of  the  antiserum. 
A  particularly  important  corroboration  of  Vaughan's  theory  is  fur- 
nished by  the  behavior  of  the  racemized  protein  of  Dakin.  Although 
soluble,  this  protein  cannot  be  attacked  by  the  digestive  proteolytic 
enzA^mes,  presumably  because  of  its  altered  configuration;  and  it  is 
non-antigenic,  presumably  because  it  cannot  be  attacked  by  the  pro- 
teases of  the  blood  and  tissues.  Likewise  it  cannot  be  metabolized, 
whether  fed  or  injected  subcutaneously.-"  Here  we  have  good  evi- 
dence of  the  fundamental  identity  of  the  three  processes,  digestion, 
metabolism,  antigenic  activity. 

As  immunity  reactions  manifest  themselves,  however,  there  are 
steps  in  the  process  besides  simple  hydrolysis  of  proteins,  even  if 
this  be  the  ultimate  goal  of  them  all.^^" 

Specificity  of  Immune  Reactions 

The  many  attempts  to  explain  the  various  reactions  of  immunity 
solel}''  on  the  basis  of  known  physico-chemical  properties  of  colloids 
all  flatten  out  when  the  striking,  characteristic,  and  often  extreme 
specificity  of  these  reactions  is  considered.  Chemical  explanations 
are  but  little  more  satisfactory.  In  enzyme  action  we  find  manj^  com- 
parable examples  of  specificity, — but  this  does  not  help,  as  the  enzymes 
are  as  mysterious  as  the  antibodies.  But  no  proposed  explanation  of 
any  of  the  reactions  incited  by  antigens  can  be  of  value  if  it  fails  to 
take  into  account  the  specificity  of  the  reactions.  We  lack  the  space 
here  to  consider  the  many  ideas  and  the  items  of  evidence  which  have 
been  advanced  concerning  this  all-important  chemical  problem,  but 
refer  the  reader  to  the  excellent  discussion  by  E.  P.  Pick.^^  The  main 
facts  at  present  available  are  the  following:  Specificity  was  at  first 
supposed  to  depend  solely  upon  biological  relationships,  for  it  was 
found  easy  to  distinguish  the  serum  of  animals  of  unlike  nature  by 
means  of  the  precipitin  and  other  reactions,  but  the  more  closely  re- 
lated the  animals  the  less  sharply  these  reactions  distinguish  them, 

"  See  Vaughan,  "Protein  Split  Products,"  Philadelphia,  1913. 

^^  Abderhalden,  "  Abwehrferinente  des  tierischen  Organism  us,"  Berlin,  1913. 

"  See  Ten  Broeck,  Jour.  Biol.  Chem.,  1914  (17),  369. 

''^"  Tadokoro  states  that  immune  sera  show  spectroscopic  differences  from 
normal  sera  .(Jour.  Infect.  Dis.,  1920  (26),  8). 

*'  Kolle  and  Wassermann's  Handbuch  d.  path.  Mikroorganismen,  1912  (1), 
685;  full  bibliography. 


166  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

until,  with  such  closely  related  animals  as  dog  and  fox,  or  man  and 
apes,  antisera  for  the  blood  of  one  react  nearly  as  well  with  the  blood 
of  the  other,  the  existing  differences  being  only  quantitative.  The 
opinion  therefore  gained  ground  that  the  specificitj'  depends  upon  some 
peculiar  biological  relationship  of  the  antigens,  and,  as  serum  proteins 
which  seem  to  be  quite  similar  chemically  but  which  are  from  un- 
related species,  are  sharply  differentiated  by  the  biological  reactions, 
that  the  specificity  must  depend  upon  something  quite  distinct  from 
ordinar}'  chemical  differences.  But  even  with  closely  related  species, 
differences  can  often  be  brought  out  by  means  of  the  process  of  satura- 
tion (which  consists  in  treating  the  antiserum  with  sufficient  quantities 
of  an  antigen  until  it  no  longer  reacts  with  additional  quantities  of 
this  antigen,  and  then  trying  its  reactive  power  with  the  other  related 
antigen  which  one  wishes  to  test). 

As  use  began  to  be  made  of  other  materials  than  serum,  and 
especially  when  more  or  less  purified  proteins  were  employed,  it  was 
found  that  within  the  tissues  of  a  single  animal  or  plant  there  might 
exist  antigens  which  were  quite  distinct  from  one  another — more  so, 
indeed,  than  some  of  the  chemically  similar  substances  of  different 
biological  origins.  Thus,  in  the  hen's  egg,  by  means  of  the  anaphy- 
laxis reaction,  I  have  been  able  to  distinguish  five  distinct  antigens, 
and  these  correspond  to  as  many  different  proteins  which  have  been 
distinguished  by  chemical  means. ^^  Also,  for  another  example,  in 
the  crystalline  lens  are  found  proteins  which  are  specific  for  lens 
proteins,  in  that  they  produce  antibodies  reacting  with  lens  proteins 
from  varied  species  of  animals,  but  not  with  the  serum  proteins  of 
the  species  from  which  the  antigenic  lens  substance  was  derived.^" 
Here  the  chemical  character  of  the  protein  is  undoubtedly  more 
significant  than  its  biological  relations.  These  and  other  observa- 
tions leave  little  room  for  doubt  that  specificity  does  depend  upon 
chemical  composition,  and  that  the  differences  in  species  as  exhibited 
by  their  biological  reactions  depend  upon  distinct  differences  in  the  chemis- 
try of  their  proteins.^^  Chemically  distinct  proteins  (e.  g.  lens  and  serum 
proteins)  of  one  animal  may  be  immunological!}'  distinct,  and  chemically 
related  proteins  of  dissimilar  species  (e.  g.  casein  from  goat  and  cow 
milk)  may  show  immunological  relationship.  Crystalline  albumin 
from  hen's  eggs  shows  no  immunological  distinction  from  that  of  ducks' 
eggs,  whereas  each  of  the  three  proteins  separable  from  horse  serum — 
euglobulin,  pseudoglobulin  and  albumin — can  be  distinguished  from 
the  other  two  by  the  anaphylaxis  reaction.  "^^  Furthermore,  it  has 
been  shown  by  Wells  and  Osborne*'^  that  a  single  pure  protein  may 
exhibit  multiple  antigenic  properties,  and  react  or  fail  to  react  with 

"  Jour.  Infec.  Dis.,  1911  (9),  147. 

•"  Krusius,  Zeit.  Irinimnit:it.,  1910  (.5),  r)99. 

"See  Wells  and  ()sl)<)ni(-,  Jour.  Infect.  Dis.,  19H)  (19),  183. 

"  Dale  and  IfarUcy,  liioclieni.  Jour.,  191C.  (10),  40S. 

"Jour.  Infect.  Di.s.,  1913  (12),  .341 


SPECIFICITY  167 

other  pure  proteins  iiecordiiifi;  to  whether  chemical  dijfferences  can 
be  demonstrated  by  recognized  analytical  methods. 

A  striking  example  of  the  existence  of  identical  antigenic  prop- 
erties in  materials  of  biologically  unrelated  origins,  is  furnished  by 
the  sheep  corpuscle  ti^|nn1ys:Ln  discovered  by  Forssner,^*  who  found 
that  man}^  different  materials,  \vhen  injected  into  rabbits,  engender  in 
the  rabbits'  serum  active  hemolytic  amboceptors  for  sheep  corpuscles. 
This  antigenic  property  has  been  demonstrated  in  the  organs  of  the 
guinea-pig,  horse,  cat,  dog,  mouse,  chicken,  turtle,  and  several  species 
of  fish,''^  although  not  exhibited  by  organs  of  many  closely  related 
species  (e.  g.  pig,  ox,  rabbit,  goose,  frog,  eel,  man,  pigeon,  rat).  It  is 
not  present  in  the  red  corpuscles  of  these  animals,  but  is  present  in 
the  corpuscles  of  the  sheep,  whose  organs  do  not  have  this  property. 
It  has  also  been  found  in  paratyphoid  and  Gartner  bacilli,  mouse 
tumors  and  sheep  spermatozoa.  Not  only  does  the  serum  of  rabbits 
thus  immunized  show  active  hemolysis  for  sheep  corpuscles,  but  if 
injected  into  the  vein  of  an  animal  whose  organs  contain  this  antigen 
there  results  a  prompt,  severe  anaphylactic  intoxication,  presumably 
through  reaction  between  the  antigen  present  in  their  tissues  and  the 
antibodies  of  the  rabbit  serum.  Furthermore,  the  antibody  can  be 
specifically  removed  from  the  immune  rabbit  serum  by  contact  with 
any  of  the  antigen-containing  tissues,  but  not  by  tissues  that  do  not 
exhibit  this  antigenic  property.  The  antigen  seems  to  remain  in  the 
tissues  when  the  fluids  are  forced  out  by  pressure,  and  Doerr  and  Pick 
believe  it  to  be  associated  with  the  nucleoproteins. 

This  series  of  observations,  which  seem  to  have  been  quite  gen- 
erally corroborated,  indicates  conclusively  that  the  immunological 
specificity  of  an  antigen  is  not  necessarily  related  to  the  biological 
specificity  of  the  living  organism  from  which  it  is  derived.  The  logi- 
cal explanation  is  that  there  may  exist  proteins  in  different  species 
which  have  chemical  resemblances  or  identity,  and  this  is  scarcely  to 
be  doubted.  We  find  identical  lipoids,  fats,  nucleic  acids,  and  carbo- 
hydrates in  different  species;  many  peculiar  types  of  proteins  show 
apparent  chemical  identity  in  different  species  (e.  g.  gelatin,  keratin) ; 
some  chemically  similar,  derived  proteins  also  seem  immunologically 
identical  or  closely  related  (e.  g.  lens  protein,  casein).  Therefore,  it 
is  highly  probable  that  many  tissue  proteins  may  be  identical  in  dif- 
ferent forms  of  animal  cells,  and  even  in  animal  and  plant  cells. 

Another  sort  of  manifestation  of  apparently  non-specific  immunity 
reactions  has  been  observed  especially  in  therapeutic  immunization.^® 
Beginning  with  the  classical  observation  of  Matthes  that  the  tuber- 
culin reaction  could  be  produced  with  deutero-albumose,  many  sim- 
ilar  non-specific   reactions   have    been    observed.     Particularly   the 

"  Review  by  Doerr  and  Pick,  Biochem.  Zeit.,  1914  (60),  257. 

"  Tsunecka,  Zeit.  Immunitat.,  1914  (22),  567. 

^«  See  review  by'Jobling,  Jour.  Amer.  Med.  Assoc,  1916  (66),  1753. 


168  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

sharp  reaction  that  follows  intravenous  injections  of  killed  typhoid 
bacilli  into  typhoid  patients  has  been  found  to  result  equally  well  if 
colon  bacilli  are  used,  or  deutero-albumose.  One  possible  explana- 
tion of  this  type  of  reaction  is  that  the  injected  substance  acts  as  a 
common  antigen,  which  causes  the  production  of  common  antibodies 
that  react  also  with  the  antigens  of  the  cause  of  the  disease.  Another 
possibility  is  that  the  foreign  protein  stimulates  the  tissues  that 
form  antibodies,  presumably  the  red  marrow,  so  that  they  produce 
not  only  antibodies  for  this  antigen,  but  also  for  the  antigens  of  the 
specific  etiologic  factor  of  the  cHsease  that  have  been  stimulating  the 
bone  marrow  previously.  Hektoen"  has  observed,  for  example,  that 
if  an  animal  that  has  previously  produced  precipitins  for  one  foreign 
protein  is  reinjected  with  a  different  protein  it  will  then  produce  pre- 
cipitins for  both  these  proteins,  and  possibly  for  other  proteins  with 
which  it  has  not  been  injected. ^^  Moreover,  the  febrile  reaction,  the 
ieucocytosis,  and  other  phenomena,  such  as  the  antiferment  index  of 
the  serum  (Jobling),^^  that  injection  of  nonspecific  protein  produces, 
may  be  responsible  for  favorably  affecting  the  disease,  rather  than 
actual  antibody  formation. 

The  opposite  type  of  phenomenon,  that  is,  non-specific  interference 
with  immunological  reaction,  is  suggested  by  the  observations  of  J.  H. 
Lewis.'"  He  found  that  small  quantities  of  one  protein  injected  into  a 
guinea  pig  together  with  or  shortly  after  large  quantities  of  another 
protein  (e.  g.,  egg  albumen  in  dog  serum)  would  not  sensitize  the 
animal,  although  a  similar  amount  injected  alone  would  always  sensi- 
tize. The  suggested  explanation  is  that  the  larger  amount  of  foreign 
protein  combines  with  so  many  of  the  available  cell  receptors  that  few 
of  the  small  number  of  sensitizing  protein  molecules  are  able  to  be 
bound  to  the  cells  and  to  stimulate  antibody  formation;  this  explana- 
tion assumes  a  certain  lack  of  specificity  on  the  part  of  the  cell  receptors. 

An  interesting  illustration  of  the  fact  that  whatever  stimulates  the 
bone  marrow  may  cause  it  to  form,  among  other  blood  elements,  spe- 
cific antibodies,  is  furnished  by  the  behavior  of  antitoxin-producing 
horses.  If  a  horse  that  has  been  immunized  to  diphtheria  toxin  is 
bled  as  much  as  possible,  it  will  be  found  to  have  regenerated  the  lost 
antitoxin  within  48  hours, ^'  even  although  the  last  immunizing  dose 
of  toxin  was  received  long  before.  Also,  it  is  stated  that  jiersons  who 
have  once  had  typhoid,  but  whose  blood  no  longer  contains  much 
agglutinin,  may  show  a  high  typhoid  agglutinin  content  when  infected 
by  some  other  organism,  or  after  any  sharji  febrile  attack.  It  is 
highly  possible  that  many  therapcnitic  agents  may  similarly  act  by 

"  Jour.  Infoc;t.  Dis.,  1917  (21),  279. 
«8  See  also  Ifemiuinii,  Udd.,  191.S  (23),  457. 

*"  See  review  of  tliis  suhject,  llarvev  Lectures,  1917  (12),  ISl;  also  Cowie  and 
Calhoun,  Anrli.  Int.  Med.,  1919  (2;{),  ()9. 
'»  Jour.  Infect.  Dis.,  1915  (17),  211. 
^'  O'Brien,  Jour.  Path,  and  Bact.,  1913  (18),  89. 


SPECIFICITY  109 

stimulatinp;  the  marrow  to  increased  formation  of  specific  antibodies,  } 
e.  g.,  arsenic,  mercury  and  other  metals,  heliotherapy,  hemorrhage^"*  ; 
or  phlebotomy,  hot  baths. 

The  other  aspect  of  specificity,  i.  e.,  the  presence  of  several  antigens 
in  a  single  organism,  each  entirely  distinct  from  other  antigens  in  the 
same  organism,  has  been  repeatedly  demonstrated.  Besides  the 
identification  of  five  distinct  antigens  in  the  hen's  egg,  mentioned 
previously,  we  have  the  repeatedly  demonstrated  individuality  of 
serum  proteins  and  milk  casein  of  the  same  animal,  and  even  the  dif- 
ferentiation of  casein  from  lactalbumin  in  the  same  milk,  as  contrasted 
with  the  common  inter-reactions  of  caseins  from  different  sources,'^ 
e.  g.,  cow  and  goat.  A  certain  but  slight  distinguishable  specificity 
may  be  observed  between  proteins  from  different  organs  of  the  same 
animal,  which  differentiation  is  still  sharper  between  the  tissue  pro- 
teins and  serum  proteins  of  the  animal."^  Sex  cells  especially  seem  to 
be  rather  distinct  immunological!}^  from  the  body  cells. '^^  Numerous 
instances  of  two  separate  proteins  from  the  same  plant  seeds  show- 
ing entirely  distinct  immunological  specificities  have  been  described. ^^ 
Although  hemoglobin  itself  seems  not  to  be  antigenic,''®  some  of  the 
most  striking  examples  of  absolute  specificity  are  furnished  by  red 
corpuscles,  which  show  readily  demonstrable  differences  between 
closely  related  individuals.  For  example,  take  the  remarkable  ob- 
servation of  Todd,*"^  who  mixed  together  isolytic  beef  sera  from  over 
60  animals,  and  then  tested  the  mixture  with  the  corpuscles  of  110 
different  cattle,  all  of  which  were  hemolyzed.  When  the  mixture  of 
sera  was  exhausted  with  the  corpuscles  of  any  one  of  the  110  cattle  it 
would  then  hemolyze  the  corpuscles  of  all  the  other  109,  but  was  ab- 
solutely without  action  on  the  corpucles  of  the  individual  with  whose 
corpuscles  it  had  been  exhausted.  This  indicates  that  the  red  cor- 
puscles of  any  individual  possess  characters  which  differentiate  them 
from  the  corpuscles  of  any  other  individual  even  of  the  same  species. 

As  satisfactory  a  conception  of  the  nature  of  specificity  as  our 
present  evidence  warrants  is  that  developed  b}'  Pick,  largelj'  on  the 
basis  of  his  own  work.  He  properly  accepts  the  influence  of  both 
the  physico-chemical  properties  and  the  chemical  composition  of  the 
colloids  concerned  in  immunity  reactions  as  determining  specificity. 
Both  these  factors  undoubtedly  come  into  play  in  determining  the 
possibility  of  interaction  of  antigen  and  antibody.  The  electric 
charges  of  the  amphoteric  colloidal  antigen  and  antibody,  and  per- 
haps also  their  surface   configuration  and  their  surface  forces,   all 

'""  See  Hahn  and  Langer.  Zeit.  Immimitat.,  1917  (26),  199. 
"  See  Versell,  Zeit.  Immunitat.,  1915  (24),  267. 
"  See  Salus,  Biochem.  Zeit.,  1914  (60),  1. 
"Kiraetz,  Zeit.  Immunitat.,  1914  (21),  150. 

"^  Wells  and  Osborne,  Jour.  Infect.  Dis.,  1911  (8),  66  et  seq.,  especially  1916 
(19),  183. 

"«  Schmidt  and  Bennett,  Jour.  Infect.  Dis.,  1919,  (25),  207. 
"  Jour,  of  Genetics,  1913  (3),  123. 


170  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

influence  their  reaction;  these  physico-chemical  factors  greatly  com- 
plicate the  possibility  of  reaction  between  two  colloids,  and  to  these 
influences  are  added  the  influence  of  the  chemical  structure  in  deter- 
mining subsequent  chemical  reactions.  It  would  seem  possible  that 
the  existence  of  all  these  factors  may  account  for  specificity,  it  being 
necessary  for  each  one  of  a  long  series  of  both  physical  and  chemical 
adjustments  to  agree  perfectly  in  order  that  reaction  may  take  place 
— 'just  as  in  a  combination  lock  one  lever  after  another  is  thrown  by 
the  proper  manipulation  of  the  dial,  and  only  when  all  the  long  series 
of  levers  is  in  just  the  proper  position  does  the  bolt  engage  and  the 
lock  open.^^ 

The  studies  of  Pick  and  his  colleagues,  amplified  somewhat  by  other 
investigations,  have  led  to  the  following  view  of  the  chemistry  of 
specificity:  There  exist  two  sorts  of  specificity  in  each  protein  mole- 
cule; one  of  these  is  easily  altered  by  simple  physical  measures,  e.  g., 
heat,  cold,  partial  coagulation,  etc.,  without  essentially  changing  the 
chemical  composition  of  the  protein.  When  so  altered  the  antigenic 
properties  of  the  protein  are  likewise  altered,  in  that  the  antibody 
it  engenders  differs  somewhat  in  the  scope  of  its  reactivity  from  the 
antibody  engendered  by  the  original  unaltered  protein;  but  the  altera- 
tion does  not  affect  the  species  characteristics  of  the  antigen.  Thus, 
a  heated  antigen  may  engender  precipitins  that  will  react  with  this 
heated  antigen,  but  not  with  similar  heated  proteins  from  other 
species  of  animals,  while  the  antibodies  engendered  by  the  same  but 
unheated  antigen  will  not  react  with  the  heated  protein. 

The  other  sort  of  specificity  is  not  so  easily  affected,  only  marked 
chemical  alterations  of  the  antigen  modifying  it,  and  this  concerns  the 
species  characteristics  of  the  antigen.  This  fundamental  species  speci- 
ficity seems  to  be  closely  related  to  the  aromatic  radicals  of  the  protein 
antigen,  for  it  is  affected  by  introducing  into  the  protein  molecules 
substances  which  are  known  to  combine  with  the  benzene  ring,  e.  g., 
iodin,  diazo  and  nitro  groups.  Proteins  thus  chemically  altered  will 
act  as  proteins  foreign  to  animals  of  the  species  from  which  they  are 
derived,  and  the  antigens  they  develop  are  devoid  of  species  specificity, 
although  quite  specific  for  proteins  like  themselves;  e.  g.,  a  nitro- 
protein  made  by  treating  rabbit  serum  protein  with  nitric  acid,  will, 
if  injected  into  even  the  same  rabbit,  cause  the  formation  of  antibodies 
which  will  react  with  this  same  nitro-protcin,  and  also  with  nitro- 
protcins  derived  from  entirely  different  species  or  even  from  plants, — 
but  it  reacts  only  with  nitro-proteins.  It  is  also  possible  to  cause 
chemical  modifications  analogous  to  the  physical  modifications  previ- 
ously mentioned,  which  change  only  the  scope  of  specificity  of  the 

"  The  "resonance  theory"  of  Traube  assumes  that  the  surface  forces  of  react- 
ing substances  must  harmonize,  just  as  the  vibration  of  one  tuning  fork  starts 
vibrations  in  another  fork  only  when  the  two  are  in  liarniony,  or  as  electromag- 
netic waves  incite  resonance  plienomena  (see  Zeit.  f.  Immunitat.,  1911  (9),  246 
and  779). 


SPECIFICITY  171 

antigen  without  altering  its  specificity  for  species.  Appreciating 
that  the  number  of  different  aromatic  radicals  in  the  protein  mole- 
cule is  not  sufficient  to  account  for  the  innumerable  manifestations 
of  specificity,  Pick  interprets  the  significance  of  these  aromatic 
radicals  as  that  of  a  central  complex  about  which  are  the  groupings 
which  determine  species  specificity.^'-*  It  is  not  merely  the  number 
and  proportion  of  amino-acid  radicals  in  the  protein  molecule  which 
determine  its  specificity,  but,  more  important  because  presenting 
greater  possibilities  for  variations,  the  arrangement  of  these  radicals 
in  the  molecule. 

Landsteiner  and  Lampl^**  have  also  carried  on  an  extensive  study 
of  the  precipitin  reactions  of  horse  serum  when  combined  with  azo- 
compounds,  with  chlorine  and  bromine  and  with  sulfonic  and  arsenic 
acids.  Their  results  confirm  the  observations  and  conclusions  of 
Obermayer  and  Pick  and  of  Wells  and  Osborne,  that  specificity  de- 
pends upon  certain  groups  within  the  protein  molecule.  They  made 
the  interesting  observation  that  if  one  derivative  of  a  protein  reacted 
with  another  sort  of  derivative,  the  position  in  the  molecule  of  the 
substituted  radicals  was  identical  or  closely  related.  That  is,  cross 
reactions  depend  on  chemical  relationships,  as  Wells  and  Osborne  also 
found  by  means  of  the  anaphylaxis  reactions,  and  the  specificity  is 
determined  by  relatively  small  portions  of  the  large  antigen  molecule. 
The  observation  that  the  location  in  the  molecule  of  definite  groups  is 
indicated  by  their  immunological  reactions  can  best  be  explained  as 
depending  on  spatial  correspondence  of  antigen  and  antibody,  just 
as  Emil  Fischer  assumed  for  the  specific  action  of  ferments  in  his 
comparison  to  "lock  and  key."  Here  again  we  get  evidence  that 
both  chemical  composition  and  spatial  relations  are  concerned  in  deter- 
mining specificity.  Presumably  there  are  also  innumerable  isomeres 
that  cannot  be  distinguished  by  our  present  methods,  which  correspond 
to  the  racial  and  individual  differences  which  are  so  obvious  and  yet 
not  to  be  detected  by  serum  reactions. 

Contemplating  the  possible  number  of  variations  in  the  arrange- 
ment of  the  amino-acids  in  a  protein  which  the  great  number  of  these 
radicals  provides,  there  is  no  diflB.culty  in  understanding  the  existence 
of  an  almost  limitless  number  of  specific  distinctions  between  proteins. 
Abderhalden,  indeed,  calculates  that  the  20  amino-acids  we  find  in 
proteins  could  form  at  least  2,432,902,008,176,640,000  different  com- 
pounds, and  this  without  including  possible  compounds  varying  in 
quantitative  relations.  A  contribution  to  the  chemical  basis  of  speci- 
ficity has  been  made  by  Kossel,''^  who  finds  certain  relations  in  the 

''Landsteiner  and  Prasek  (Zeit,  Immunitat.,  1913  (20),  211),  however,  state 
that  alteration  of  proteins  by  simply  treating  them  with  acid  alcohol  also  causes 
them  to  lose  their  species  specificity,  and  this  without  anj'-  substitution  in  the 
aromatic  radicals  of  the  proteins.  This  observation  throws  doubt  on  the  hypothe- 
sis of  Pick  that  the  aromatic  radicals  are  the  essential  center  of  species  specificity. 

«»  Biochem.  Zeit.,  1918  (86),  343. 

«i  Zeit.  physiol.  Chem.,  1913  (88),  163. 


172  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

proportions  and  groupings  of  the  scanty  number  of  amino-acids  that 
make  up  the  protamines  and  histones  of  sperm  to  be  characteristic 
of  the  sperm  of  certain  species  and  famihes. 

In  the  subsequent  discussion  of  the  various  reactions  of  immunity 
the  subject  of  specificity  will  receive  further  consideration.  Of  these 
reactions,  one  of  the  simplest  and  most  studied  is  that  of 

TOXINS  AND  ANTITOXINS 

In  the  preceding  chapter  on  the  bacteria  and  their  products  the 
nature  of  the  true  toxins  was  defined,  and  attention  was  called  to  the 
fact  that  one  of  their  most  important  characteristics  is  that  immuniza- 
tion of  animals  against  them  leads  to  the  accumulation  in  the  blood 
of  substances  capable  of  neutralizing  their  poisonous  action.  Such 
true  toxins  are  produced  especially  by  the  diphtheria  bacillus  and  the 
tetanus  bacillus;  also,  but  less  strikingl}^,  by  B.  pyocyaneus,  B.  botu- 
linus,  pathogenic  gas  bacilli,  dysentery  bacilli,  and  possibly  by  a  few 
others.  In  addition  to  these,  numerous  bacteria  produce  hemolytic 
poisons  w^hich  seem  to  have  properties  similar  to  the  toxins;  and  there 
are  also  toxins  produced  b}^  plants  (abrin,  ricin,  crotin,  and  mushroom 
poisons)  and  by  animals  (snake  venom,  scorpion  and  spider  toxin,  and 
eel  serum).  Against  all  of  these,  true  antitoxins  may  be  obtained  by 
the  immunization  of  animals. 

Ehrlich's  Conception  of  Toxins  and  Antitoxins. — According 
to  Ehrlich's  theory,  the  action  of  toxins  upon  cells  is  purely  ^hejuinaL 
A  toxin  unites  with  a  cell  because  some  chemical  grovip  in  the  molecule 
of  toxin  has  a  chemical  affinity  for  some  particular  group  in  the  cell 
protoplasm.  For  convenience  in  description  names  have  been  given 
to  these  groups;  the  group  of  the  toxin  that  combines  with  the  cell 
has  been  called  the  haptophorous  grouD,  or  haptophore,  while  the 
group  in  the  protoplasm  that  combines  with  the  toxin  is  known  as 
the  receptor.^^  It  has  been  found  that  after  being  kept  for  some 
time,  or  when  placed  under  certain  unfavorable  conditions,  the  toxin 
loses  its  poisonous  properties  without  losing  its  power  to  combine 
with  cells,  as  shown  by  the  fact  that  immunization  with  such  altered 
toxin  gives  rise  to  the  formation  of  antitoxin.  Therefore  it  is  not  the 
haptophore  that  causes  the  harm  to  the  cell,  but  there  must  be  some 
other  groups  with  this  particular  function.  To  the  group  that  pro- 
duces the  harm  the  name  toxophorc  is  given.  If  all  tlu>  receptors  of 
a  cell  are  coni])ined  by  toxin  molecules  that  have  lost  their  toxophore 

*^  Ehrlich  has  used  certain  diaKrams  to  illustrate  these  various  groups  and 
their  relations  to  the  cells  and  to  one  another,  which  arc  generally  used  in 
exi)lairiiiig  his  thef)ry.  From  a  teaching  standpoint  they  have  seemed  to  be 
umicsiralilc,  in  that  the  student  soon  comes  to  ascril)e  piiysical  properties  and 
ai)i)caranc('s  to  what  should  he  considered  as  oliemical  coml)inations.  The 
toxopliorc  grouj)  Ix'comes  "the  hlack  fringed  end  of  the  toxin."  etc.  To  one 
accustomed  to  thinking  in  chemical  terms  there  is  no  diiiiculty  in  following  the 
literature  and  understanding  the  reactions  as  chemical  reactions,  which  they  are. 


TOXINS  AND  ANTITOXINS  173 

p;r()up  (toxoid  is  the  muuc  given  to  such  altei-ed  toxins),  tiie  cell  can- 
not then  be  injured  by  the  corresponding;  active  toxin,  showing  that 
the  toxin  nuist  first  become  united  to  a  cell  receptor  by  its  haplophorc 
group  before  the  toxophore  group  can  cause  an  injury. 

Animals  that  are  naturally  immune  to  toxins  may  owe  their  im- 
munity to  the  fact  that  their  vital  tissues  contain  no  substances  with 
a  chemical  affinity  for  the  toxin,  and  hence  the  toxin  cannot  unite 
with  them  to  cause  harm.  (In  Ehrlich's  terminology,  the  cells  con- 
tain no  receptors  for  the  toxin.)  The  toxin  may  not  combine  with 
any  tissue  element  at  all  in  such  immune  animals,  and  may  circulate 
for  some  time  harmlessly  in  the  blood,  or  it  ma}^  combine  with  some 
organ  where  it  does  little  harm,  e.  g.,  tetanus  toxin  is  said  to  combine 
chiefly  in  the  liver  of  some  animals,  and  therefore  it  does  not  harm 
their  nervous  system. 

According  to  this  theory,  the  antitoxin  consists  of  cell  receptors 
that  have  been  produced  in  excess  and  secreted  by  the  cells  into  the  blood. 
In  the  blood  they  combine  with  any  toxin  that 'may  have  been  intro- 
duced, and  by  saturating  its  affinities  render  it  incapable  of  uniting 
with  the  cells.  As  the  toxin  harms  cells  only  after  it  has  been  chemi- 
cally united  to  them,  it  is  rendered  harmless  when  its  affinities  for  the 
cell  (the  haptophore  groups)  are  saturated  by  cell  receptors  in  the  blood 
stream.  The  process  of  immunization  consists  in  injuring  the  body 
cells  to  such  a  degree  that  they  are  stimulated  to  regenerate  the 
receptor  groups  with  which  the  toxin  combines;  these  receptor  groups 
are  produced  in  excess,  and  not  only  replace  those  combined  by  the 
toxins,  but  the  excessive  groups  escape  free  into  the  blood.  Hence 
the  serum  of  an  immunized  animal  is  antitoxic  because  it  contains  free 
cell  receptors  that  can  unite  with  the  toxin.  An  important  point  is 
that  the  receptors  liberated  by  all  animals  which  have  been  immunized 
wdth  a  given  toxin  seem  to  be  the  same — horse  serum,  or  sheep  serum, 
or  goat  serum  will  neutralize  diphtheria  toxin  if  the  animals  have  been 
made  immune  to  this  toxin;  and,  furthermore,  their  serum  when  intro- 
duced into  the  body  of  an  entirely  different  animal,  e.  {7.,  a  guinea-pig, 
will  neutralize  diphtheria  toxin  within  its  body.  Equally  important 
is  the  fact  that  the  antitoxin  for  one  toxin  will  not  neutralize  any  other 
toxin;  e.  g.,  diphtheria  antitoxin  will  not  neutralize  tetanus  toxin,  or 
conversely.  This  means  that  diphtheria  toxin  is  attached  to  chemical 
groups  of  the  body  cells  (receptors)  which  are  quite  difTerent  from  the 
groups  to  which  tetanus  toxin  unites,  and  hence  different  receptors 
are  thrown  out  in  immunizing  against  each.  True  toxins  have  been 
designated  monovalent  antigens^  since  animals  immunized  with  a  puri- 
fied toxin  produce  onl}^  the  one  antibody,  the  antitoxin,  whereas  many 
protein  antigens  produce  precipitins,  Ij^sins,  agglutinins  and  other 
antibodies;  presumably  this  is  because  of  the  relatively  small  size  of 
the  toxin  molecule,  which  limits  the  number  of  its  antigenic  radicals 
(Pick).     Or  it  may  well  be  that  the  immune  body  for  antitoxin  is 


174  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

quite  different  from  the  antibody  or  antibodies  resulting  from  immuni- 
zation with  non-toxic  protein  antigens,  for  there  is  some  reason  to 
believe  that  the  several  types  of  reactions  that  may  be  accomplished 
with  the  serum  of  animals  immunized  to  foreign  proteins  or  cells  all 
depend  on  one  single  antibody,  which  accomplishes  the  destruction  of 
the  antigen  by  sensitizing  it  to  the  enzymes  of  the  blood  and  tissues. 
The  neutralization  of  toxin  by  antitoxin  is  believed  by  many  in- 
vestigators to  be  a  chemical  process,  which  occurs  as  well  in  the  test- 
tube  as  in  the  body.  It  seems  to  occur  according  to  the  laws  of  definite 
proportion,  sl  given  amount  of  antitoxin  neutralizing  a  proportionate 
amount  of  toxin  under  equal  conditions  (hence  the  toxin  is  not  de- 
stroyed by  antitoxin  through  a  ferment  action,  as  was  at  first  suggested). 
.Neither  the  toxin  nor  the  antitoxin  is  destroj^ed  in  the  process  of  neu- 
tralization, as  has  been  proved  by  suitable  experiments,  but  they  appear 

/to  be  chemically  united  to  each  other,  as  any  two  large  molecules  may 
be.  Pick  and  Schwarz  believe  that  the  union  of  toxin  and  antitoxin 
takes  place  in  two  steps — first,  colloidal  adsorption,  and  then  the  specific 
reaction. ^^  There  is  some  question  as  to  whether  the  union  with  anti- 
toxin completes  the  neutralization  of  the  toxin,  or  whether  there  is  then 
necessary  a  further  destruction  of  the  toxin  in  the  body.  But  whether 
necessary  or  not,  such  further  destruction  does  take  place.     Neutrali- 

j  zation  occurs  more  rapidly  under  the  influence  of  warmth,  and  more 
slowly  in  the  cold;  and  it  is  more  rapid  in  concentrated  than  in  dilute 
solutions,  just  as  with  ordinary  chemical  reactions.  It  is  said  that  it 
requires  two  hours  for  tetanus  toxin  to  be  completelj^  combined  with 
the  corresponding  quantity  of  antitoxin  at  37°.  According  to  Arrhe- 
nius  and  Madsen,  reaction  of  antitoxin  upon  toxin  is  accompanied  by 
the  liberation  of  much  heat — 6600  calories  per  gram  molecule,  or  about 
half  as  much  as  is  set  free  by  the  action  of  a  strong  acid  upon  a  strong^ 
base.^^  Union  of  toxin  and  antitoxin  causes  no  change  in  the  surface 
tension  of  the  fluid  in  which  the  reaction  occurs  (Zunz),*^  and  the 
neutral  toxin-antitoxin  compound  (diphtheria)  is  not  absorbed  by  ani- 
mal charcoal,  which  absorbs  each  of  the  constituents  when  free.  The 
physico-chemical  studies  of  the  reaction  between  tetanolysin  and  its- 
antibody  gave  results  which  led  Arrhenius  to  conclude  that  in  the 
reaction  there  are  formed  from  one  molecule  of  toxin  and  one  molecule 
of  antitoxin,  two  molecules  of  the  reaction  products  (analogous  to  the 
reaction  between  alcohol  and  acid  which  yields  one  molecule  of  ester 
and  one  of  water).     In  general,  the  union  of  toxin  and  antitoxin  is 

"Also  von  Krogh  (Zeit.  f.  Hyg.,  1911  (68),  251).  Bordet,  Biltz,  and  others 
look  upon  the  neutralization  of  toxin  as  an  adsorption  process  entirely. 
g  **  Literature  of  chemical  and  jihysical  reactions  of  toxin  and  antitoxin  given 
■  by  Zangger,  Cent.  f.  Bakt.  (ref.),  1905  (80),  238;  Arrhenius,  "Ininiuno-cheni- 
listry,"  1907  and  "(Quantitative  Laws  in  Biological  Chemistry,"  London,  1915;. 
I  also  review  in  Zeit.  Chemother.,  Kef.,  1914  (3),  157;  Oppenheimer  and  IMichaelis, 
iHandbuc'li  der  Biochemie,  Vol.  II  (1). 

^  85  Bull.  Acad.  Royal  i\Ied.  Belg.,  1911;  also  Bertolini,  Biochem.  Zeit.,  1910  (28), 
60. 


ANTITOXINS  175 

dissociated  by  acids. ''^  On  dilution  of  a  neutral  toxin-antitoxin 
mixture,  a  certain  amount  of  dissociation  seems  to  occur,  but  there  is 
opposition  to  the  view  that  the  law  of  mass  action  applies  to  the  re- 
action between  toxin  and  antitoxin.  If  toxin  is  added  to  antitoxin  in 
several  fractions,  with  some  interval  of  time  between  each  addition, 
the  final  mixture  is  much  more  toxic  than  if  the  same  quantities  of 
toxin  and  antitoxin  were  put  together  at  one  time.  This  phenomenon 
is  commonly  referred  to  as  the  Danysz  effect,  and  indicates  that  the 
toxin-antitoxin  union  is  physical  rather  than  chemical,  for  it  seems  to 
be  quite  analogous  to  such  a  phenomenon  as  the  taking  up  of  more 
dye  by  several  pieces  of  blotting  paper  added  in  series  to  a  dye  solution, 
than  by  the  same  amount  of  paper  added  in  one  piece. 

There  is  no  relation  between  antitoxins  and  enzymes.  The  anti- 
toxin acts  quantitatively,  and  produces  no  detectable  alteration  in  the 
toxin,  or  in  any  other  substance,  as  far  as  we  know.  It  also  has  but 
one  functioning  group  (haptophore),  the  one  with  which  it  combines 
with  the  toxin;  whereas  both  toxins  and  enzymes  seem  to  have  two 
functionating  groups,  one  which  unites  with  the  cell  or  substance  that 
is  to  be  attacked,  the  other  which  produces  the  chemical  changes. 
But  there  is  evidence  that  union  with  antitoxin  or  fixed  receptors  pre- 
pares the  toxin  for  its  disintegration,  which,  presumably,  is  then 
accomplished  by  enzymatic  action  as  with  other  antigens. 

Chemical  Nature  of  Antitoxins*^ 

This  is  as  entirely  unknown  as  is  the  nature  of  the  toxins.  In- 
vestigation of  antitoxic  serum  (principally  diphtheria  antitoxin)  has 
shown  that  the  antitoxic  properties  are  closely  related  to  the  serum 
globulin,  which,  however,  by  no  means  proves  that  antitoxin  is  serum 
globulin  or  any  other  sort  of  protein.  According  to  Ehrlich's  theory, 
antitoxin  consists  of  free  cell  receptors,  and  these  receptors  are  pre- 
sumably simple  chemical  groups  which  may  be  but  a  part  of  a  larger 
molecule,  or  they  may  be  entire  protein  molecules.  In  any  event 
they  behave  as  colloids;  moving  toward  the  cathode  in  an  electrical 
field, ^^  diffusing  little  or  not  at  all,  their  reaction  curve  resembling 
more  an  absorption  curve  than  the  reaction  curves  of  crystalloids,  and 
being  influenced  by  all  conditions  that  influence  colloids.  Whether 
the  receptor  groups  are  secreted  in  a  free  condition  in  antitoxin*^  for- 
mation, or  combined  in  a  large  molecule,  is  unknown. 

By  saturating  serum  with  magnesium  sulphate,  or  half  saturation 
with  ammonium  sulphate,  three  chief  groups  of  proteins  can  be  pre- 

8«  Morgenroth  and  Ascher,  Cent.  f.  Bakt.,  1911  (59),  510. 

'^  Review  and  bibliography  given  by  Crawford  and  Foster,  Amer.  Jour.  Phar- 
macy, 1918  (90),  765. 

88  According    to  Field  and  Teague   (Jour.   Exper.   Med.,    1907   (9),   86)  both  \ 
toxin  and  antitoxin  move  towards  the  cathode,  which  is  opposed  to  the  theory 
that  this  reaction  is  simply  one  of  oppositely  charged  colloids.      (See  also  Bechhold,  I 
Munch,  med.  Woch.,  1907  (54),  1921.)  ' 


176  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

cipitated  and  isolated. ^^  These  are  fibrinogen,  euglohulin  (true  glob- 
ulin), and  -pseudo-globulin  (soluble  in  water).  Pick^"  found  that 
the  precipitate  obtained  by  36  per  cent,  volume  saturation  with  am- 
monium sulphate  contained  no  antitoxin;  the  antitoxin  came  down 
in  the  precipitate  obtained  on  raising  the  strength  from  above  38 
per  cent,  to  46  per  cent."  According  to  Pick,  in  horse  serum  the  anti- 
toxin is  associated  with  the  pseudo-globulin, ^^  and  Gibson  and  Banzhaf 
found  that  the  blood  of  horses  immunized  to  either  diphtheria  or  tetanus 
toxin  shows  a  marked  increase  (40  to  114  per  cent.)  in  serum  globulin, 
varying  somewhat  according  to  the  antitoxin  content,  the  more  solu- 
ble globulins  being  most  increased.  At  the  same  time  the  serum  al- 
bumin and  euglobulin  content  decreases  in  proportion,  while  the 
fibrinogen  shows  no  characteristic  alterations.^^  Mej^er^"*  and  his 
colleagues,  however,  find  in  their  study  of  the  blood  proteins  during 
immunization,  that  the  proportion  of  globulins  increases  according  to 
the  severity  of  the  intoxication,  and  not  in  any  definite  relation  to  the 
degree  of  immunity  or  antitoxin  production.  The  average  antitoxic 
horse  serum  contains  12  per  cent,  albumin,  78  per  cent,  of  soluble  glob- 
ulin containing  antitoxin,  10  per  cent,  euglobulin.  By  heating  12 
hours  at  57°  a  considerable  part  of  the  soluble  globulin  becomes 
insoluble,  without  a  corresponding  loss  of  antitoxin  (Banzhaf) . 

The  relation  of  antitoxins  to  proteins  has  also  been  investigated 
by  permitting  digestive  enzymes  to  act  on  antitoxic  serum.  Pick  di- 
gested the  antitoxin-containing  globulin  of  horse  serum  for  several 
days  with  trypsin;  after  five  days,  when  part  of  the  protein  was  still 
not  digested,  the  antitoxin  was  but  little  impaired  in  strength;  after 
nine  days,  when  most  of  the  protein  was  digested,  the  antitoxin  had 
lost  two-thirds  of  its  strength.  This  indicates  a  considerable  resist- 
ance of  antitoxin  to  trypsin,  but  also  shows  that  it  is  affected  in  much 
the  same  way  as  the  globulin  (which  is  itself  very  resistant  to  trypsin) 
and  therefore  is  presumably  of  similar  nature.  Antitoxin  seemed  to 
be  much  more  rapidly  destroyed  by  pepsin-HCl  digestion  than  by 
trypsin,  in  which  respect  it  again  resembles  the  serum  globulin. ^^ 

*^  See  rcsum6  by  Gibson,  Jour.  Biol.  Chem.,  1905  (1),  161;  Gibson  and  Banzhaf, 
Jour.  Exper.  Med.,  1910  (12),  411. 

90  Hofmeister's  Beitr.,  1901  (1),  351. 

91  Gibson  and  Collins  (Jour.  Biol.  Chem.,  1907  (3),  233)  question  the  reliabil- 
ity of  some  of  Pick's  results,  and  repudiate  the  salt  fractionation  method  of  clas- 
sifying proteins. 

92  Miss  Homer  found  tetanus  and  diphtheria  antitoxin  associated  with  the 
pseudoKlobulins,  but  the  antibodies  in  antidj^sentcry  and  antimeningococcus 
serum  were  chiefly  in  the  euglobulin  fraction  (Jour.  Physiol.,  1918  (52),  xxxiii). 

93  During  immunization  the  antitryplic  power  of  the  horse  serum  increases 
with  the  antitoxin  increase  (Krause  and  Klug.  Berl.  klin.  Woch.,  190S  (45),  1454.) 

9'  Jour.  Exp.  Med.,  1916  (24),  515;  1917  (25),  231;  Jour.  Infect.  Dis.,  1918  (22),  1. 

9'' P,erg  and  Kclser  (Jour.  Agric.  Ri-s.,  1918  (13),  471)  found  that  trypsin  and 
pepsin  destroy  tlie  antitoxin  and  scrum  proteins  at  about  the  same  rate,  and  their 
failure  to  observn;  "significant  cliemical  ciuinges"  in  the  proteins  of  serum  acted 
upon  by  weak  acid  or  alkali  that  slowly  inactivates  antitoxin,  does  not  seem  to 
warrant  tlieir  deduction  that  antitoxin  is  non-protein.  See  also  Crawford  and 
Andrus,  Amer.  Jour.  Pharm.,  1917  (89),  158. 


ANTITOXINS  111 

In  favor  of  tho  view  that  antitoxin  is  a  definite  protein  body  is 
also  the  fact  that  it  is  not  carried  down  in  indifferent  precipitates,  as 
are  the  enzymes,  but  conies  down  always  in  a  certain  fraction  of  the 
protein  precipitates,  e.  g.,  we  can  precipitate  all  the  serum  albumin 
from  an  antitoxic  serum,  and  it  does  not  carry  down  with  it  any  of 
the  antitoxin.  Another  important  point  has  been  brought  out  by 
Ai-rhenius  and  IMadsen,^'''  who  determined  approximate^^  the  molecu- 
lar weight  of  toxin  and  antitoxin  by  means  of  their  rate  of  diffu- 
sion, and  found  that  the  toxin  (diphtheria  toxin  and  tetanolysin) 
diffused  ten  or  more  times  as  rapidh'  as  the  corresponding  antitoxin. 
Gelatin  filters  also  hold  back  antitoxin  and  let  toxin  pass  through, 
and  toxins  diffuse  into  cells  which  seem  to  be  impermeable  for  the  anti- 
toxin. This  indicates  that  the  antitoxin  molecules  are  much  larger 
than  the  toxin  molecules,  agreeing  with  the  idea  that  antitoxin  is  of 
protein  nature  and  that  toxin  either  is  not  protein  or  is  smaller  than 
most  protein  molecules. 

Taken  altogether,  the  evidence  indicates  a  closer  resemblance  of 
antitoxins  to  proteins  than  has  been  show^n  for  the  toxins,  and  all 
attempts  to  separate  antitoxins  from  proteins  have  so  far  failed. 

Antitoxins  are  retained  to  greater  or  less  extent  by  porcelain 
filters,  do  not  pass  through  dialyzing  membranes  readily,  and  are  in 
general  easily  destroyed  by  chemical  and  physical  agencies,  although 
much  less  so  than  are  most  toxins.  Heating  to  60°-70°  injures,  and 
boiling  quickly  destroys  them,  although  like  the  enzymes  and  the  pro- 
teins, they  resist  dry  heat  to  140°,  and  also  extremely  low  temperature, 
without  change.  Putrefaction  of  the  serum  destroys  the  antitoxins 
(Brieger)."  They  can- be  preserved  for  a  very  long  time  when  dried 
completely,  but  in  the  serum  they  gradually  disappear,  especially  if 
exposed  to  light  and  au*.  Acids  and  alkahes  destroy  antitoxins, 
acids  being  the  more  harmful  in  low  concentrations.  Like  the  enzymes, 
antitoxins  are  destroyed  hj  ultra-violet  ra3-s.  They  are  destroyed  in 
the  alimentar}'  tract,  without  appreciable  absorption,  except  in  the 
case  of  new-born  animals  sucking  mothers  whose  blood  and  milk 
contain  antitoxin. ^^  When  subcutaneousl}'-  injected,  antitoxin  soon 
disappears  from  the  blood;  part  may  be  bound  to  the  tissues,  part  may 
be  destroyed,  since  only  traces  appear  in  the  urine.  It  resists 
autolysis. ^^ 

"  Festskrift  Statens  Serum  Institut,  1902. 

"  Behring  states  that  tetanus  antitoxin  resists  putrefaction. 

98  Romer  and  Much,  Jahrb.  f.  Kinderheilk.,  1906  (63),  684;  McCIintock  and 
King  (Jour.  Infect.  Dis.,  1906  (3),  701)  found  appreciable  absorption  of  antitoxin 
when  digestion  was  impaired  bj^  drugs.  Full  review  of  literature  on  transmission 
of  antibodies  from  mother  to  offspring  given  bv  Famulener,  Jour.  Infect.  Dis., 
1912  (10),  332;  Heurlin,  Arch.  Mens.  Obs.  et  Gvn.,  1912  (1),  497. 

"  Wolff-Eisner  and  Rosenbaum,  Berl.  klin.  Woch.,  1906  (43),  945. 


12 


178  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

AGGLUTININS  AND  AGGLUTINATION^ 

The  relation  of  agglutination  of  bacteria  by  the  serum  of  immunized 
animals  to  their  immunity  is  not  known,  for  it  is  not  established  that 
agglutination  helps  in  the  defensive  reaction. ^  Agglutinated  bacteria 
seem  not  to  be  severelj'  injured  by  the  process,  and  can  grow  vigorously 
in  agglutinative  serum.  Possibly  agglutination  favors  phagoc3'tosis 
and  lessens  dissemination  of  the  infecting  organisms,  but  it  is  not 
generally  considered  that  the  influence  on  the  com-se  of  infection  is 
great. ^  Agglutination,  therefore,  may  be  looked  upon  as  an  incident 
in  the  infection,  rather  than  as  a  definite  method  of  resistance,  and  it  is 
equally  well  produced  by  immunizing  with  foreign  cells  or  any  foreign 
protein  masses  of  suitable  size  which  contain  soluble  antigens. 

For  the  production  of  agglutination  it  is  necessary  that  the  cell 
contain  an  antigen  (agglutinogen)  which  has  an  affinity  for  the  specific 
constituent  of  the  serum,  agglutinin.  Normal  serum  may  contain 
agglutinin;'*  e.  g.,  typhoid  bacilli  are  sometimes  agglutinated  by  normal 
serum,  even  when  it  is  diluted  thirty  times,  but  by  immunization  this 
property  can  be  greatly  increased  until  agglutination  may  be  obtained 
with  dilutions  as  high  as  one  to  a  million.  Whether  normal  agglutinins 
are  essential^  different  from  immune  agglutinins  is  not  known. °  Many 
protein  solutions,  especially  extracts  of  plant  tissues  and  leguminous 
seeds,  cause  marked  non-specific  hemagglutination."  Likewise,  bac- 
terial extracts  may  agglutinate  red  corpuscles.^  In  immunization  the 
agglutinogen,  which  is  probably  an  intracellular  protein,  acts  as  a  stimu- 
lator to  the  formation  of  the  specific  agglutinin.  Hence,  when  we 
inject  either  extracts  of  cells  or  entire  cells,  we  secure  agglutinins,  for 
the  agglutinogens  are  liberated  from  the  cells  upon  their  disintegra- 
tion.    In  erythrocytes  ^he  agglutinogen  seems  to  be  in  the  stroma.^ 

We  can  obtain  agglutinins  against  nearly  all  bacteria,  including 
non-pathogenic  forms,  but  in  varying  strengths.  Agglutinins  are 
found  in  the  blood  stream  in  the  highest  concentrations,  but  they  are 

^  Bibliography  given  by  Miiller,  Oppenheimer's  Handbuch  der  Biochemie,  1909 
(II  (1),  592:  Landsteiner,  ibid.,  p.  428;  Paltauf,  Kolle  and  Wassermarin's  Hand- 
buch., 1913  (II),  483. 

^  Bull,  however,  would  ascribe  much  importance  to  agglutination  of  bacteria 
for  their  removal  from  the  circulation  (Jour.  Exj).  Med.,  1915  (22),  48-4).  P\ijimoto 
(Jour.  Immunol.,  1919  (4),  G7)  also  attributes  to  agghitinins  the  jKiwer  to  impair 
the  glucolytic  action  of  B.  colt,  but  there  is  no  evidence  in  his  experiments  that 
it  is  agglutinin  rather  than  some  other  serum  component  that  is  responsible.  On 
the  otiicr  hand  Zironi  (Atti  accad.  Lincei,  1917  (2G),  19)  found  that  agglutination 
does  not  modify  reproductive  or  biochemical  activities  of  bacteria. 

^  IJiaizot  (0.  R.  Soc.  Biol.,  1918  (81),  350)  states  that  it  is  possible  to  modify 
typhoid  baciUi  l)v  treating  them  with  nitric  acid,  hydroquinone  or  bj''  heat,  so 
that  they  will  jjrodiicf!  immunitj'  without  producing  agglutinins. 

*  Even  cold  blooded  animals  may  have  normal  agglutinins  for  bacteria  and 
mammalian  corjjuscles  (.see  Takeiiouchi,  Jour.  Inf.  Dis.,  1918  (23),  393,  415. 

*.See  Andrejew,  Arb.  kaiserl.  (iesuntlhtsamt.,  1910  (33),  84. 

«  Mendel,  Arch.  Fisiol.,  1909  (7),  KiS. 

'  Fiikuhara,  Zeit.  Immunitat.,  1909  (2),  313. 

8  Chyosa,  Arch.  f.  Ilyg.,  1910  (72),  191. 


AGGLUTINATION  17'.) 

also  present  in  the  various  organs,  and  to  greater  or  less  extent  in 
the  other  body  fluids,  excepting  usually  the  spinal  fluid  (Greer  and 
liecht).''*  The  place  of  their  founation  is  unknown,  but  they  can  be 
formed  by  spleen  tissue  grown  in  artificial  cultures.'"  vSince  bacteria 
contained  within  a  collodion  sac  iinplantcfl  in  an  animal  give  rise  to 
the  production  of  agglutinins,  it  is  evident  that  the  agglutinogens 
are  diffusible  to  some  extent,  at  least,  through  collodion.  Old  cul- 
tures of  bacteria  contain  free  agglutinogens,  probably  liberated  from 
disintegrated  cells,  and  filtrates  of  such  cultures  will  neutralize  ag- 
glutinins, showing  both  that  the  agglutinogens  arc  filtei-al)le,  and 
that  the  reaction  of  agglutination  is  a  chemical  one  and  not  dependent 
upon  the  presence  of  cells.  Agglutinogens  are  said  to  pass  through 
dialyzing  membranes,  while  agglutinins  do  not,  so  it  is  evident  that  the 
agglutinogen  is  of  smaller  molecular  dimensions  than  the  agglutinin, 
just  as  toxin  molecules  are  smaller  than  antitoxin  molecules.  Agglu- 
tinogens are  not  destroyed  by  formalin,  heat,  or  ultraviolet  rays  in 
concentrations  sufficient  to  kill  the  bacteria  containing  them.'' 

Just  what  constituent  of  the  bacteria  acts  as  the  stimulus  to  the 
production  of  the  agglutinin  is  unknown.  Apparently',  there  are 
at  least  two  bacterial  substances  with  this  property,  one  of  which 
seems  not  to  be  a  protein,  since  it  is  soluble  in  alcohol,  gives  no  biuret 
reaction  and  resists  temperatures  up  to  165°.  The  other  gives  all 
protein  reactions,  and  is  destroyed  by  heating  to  62°.  We  consider, 
therefore,  that  there  are  two  agglutinogens  in  the  bacterial  cell,  one, 
thermostable,  the  other,  thermolabile.  The  difference  in  the  func- 
tion of  these  two  agglutinogens  is  still  a  matter  of  dispute.  Likewise, 
the  question  as  to  whether  they  occur  in  the  membrane  or  within  the 
bacterial  cell  is  still  open,  but  Craw  found  that  the  insoluble  residue 
of  crushed  typhoid  bacilli,  after  being  washed  free  of  all  soluble  con- 
stituents, was  but  slightly  agglutinated  by  active  serum;  therefore, 
the  agglutinogens  are  probably  soluble  intracellular  substances.  Stuber 
holds  that  bacterial  agglutinogens  are  lipins.'^ 

Properties  of  Agglutinins. — Like  most  of  the  other  immune  substances,  agglu- 
tinins are  precipitated  out  of  the  serum  in  the  globulin  fraction.  All  attempts 
to  separate  them  from  proteins  have  been  unsuccessful.  Stark'^  found  that 
trypsin  does  not  attack  the  agglutinins  readily,  corresponding  to  the  resistance 
of  the  serum  globulins  to  this  enzyme;  alkaline  papayotin  solution  destroys  them 
slowly,  while  pepsin  acts  more  rapidly.  Alkalies  are  destructive  even  when  quite 
dilute,  while  acids  are  much  less  harmful.  The  temperature  resistance  of  agglu- 
tinins seems  to  be  variable,  plague  agglutinin  being  destroyed  at  56°,  while  purified 
typhoid  agglutinin  may  resist  80°-90°;  most  agglutinin  serums  lose  their  activity 
at  60°-65°.  The  rate  of  reaction  of  agglutinins  increases  with  the  temperature, 
as  long  as  this  is  not  high  enough  to  injure  the  reacting  substances.^'*  They  are 
not  precipitated  by  specific  precipitins,  but  are  readily  absorbed  by  charcoal. 

3  Jour.  Infect.  Dis.,  1910  (7),  127. 
>"  Pryzgode,  Wien.  klin.  Woch.,  1913  (26),  841. 
^1  Stassano  and  Lematte,  Compt.  Rend.  Acad.  Sci.,  1911  (152),  623. 
»2  Biochem.  Zeit.,  1916  (77),  388;  also  Bauer,  Biochem.  Zeit.,  1917  (83),  120. 
1'  Inaug.  Dissert.,  Wiirzburg,  1905. 
1*  Madsen,  et  al,  Jour.  Exper.  Med.,  1906  (8),  337. 


"^ 


180  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

The  observations  of  Bond'^  suggest  that  they  may  become  physically  bound  to 
other  colloids  within  the  body. 

The  structure  of  the  agglutinins  (in  the  Ehrlich  theory)  is  similar  to  that  of  the 
toxin;  i.  e.,  there  is  a  haptophore  group  by  which  they  combine  with  the  aggluti- 
nogen, and  a  toxophore  group  by  which  they  produce  the  changes  that  cause  agglu- 
tination. The  agglutinogen  is  probably  related  to  the  antitoxins  in  structure, 
having  a  single  haptophore  to  unite  with  the  agglutinin.  By  degeneration  of  the 
toxophorous  group  of  the  agglutinin,  agghitinoids  may  be  formed.  It  is  believed 
that  agglutinins  are  cell  receptors,  which  have  a  group  with  a  chemical  affinity 
for  the  agglutinogen  of  the  bacterial  protoplasm,  and  also  another  group  which 
brings  about  the  agglutination.  Thej'  are,  therefore,  more  complex  than  the 
simple  receptors  that  unite  with  toxins,  and  are  called  receptors  of  the  second  order. 
According  to  Ohno^®  the  reaction  of  agglutinin  and  antigen  is  in  constant  propor- 
tions, and  seems  to  be  a  chemical  rather  than  a  physical  reaction.  Coplans^^ 
finds  this  reaction  associated  with  an  increase  in  conductivitj-  in  the  solutions,  but 
whether  this  depends  upon  the  agglutinin  reaction  itself,  or  upon  associated 
processes,  is  questionable. 

Agglutinated  bacteria  can  be  again  separated  from  one  another  by  the  action 
of  organic  and  inorganic  acids,  alkalies,  acid  salts,  and  bj"-  heating  to  70''  or  75°. 
and  after  once  being  separated  they  cannot  be  reagglutinated  by  fresh  serum. i* 

The  Mechanism  of  Agglutination. — This  has  been  a  fruitful 
field  of  research,  in  which  the  application  of  physical  chemistry  has 
been  very  profitable.  At  first  it  was  believed  that  the  clumping  was 
brought  about  by  loss  of  motility,  until  it  was  found  that  non-motile 
bacilli  were  equally  affected.  Similarly,  the  hypothesis  of  adhesion  of 
the  fiagellse  was  disposed  of.  Gruber^^  and  others  supposed  that  a 
sticky  substance,  "  glabrificin,"  was  absorbed  from  the  serum  by  the 
bacilli,  which  caused  them  to  adhere  on  contact  with  one  another; 
but  this  does  not  explain  the  flocking  together  of  non-motile  bacilli. 
Paltauf  considered  that  the  specific  precipitin  (see  next  section)  pro- 
duced by  immunization  carried  the  bacilli  down  in  the  precipitate 
formed,  and  there  is  reason  to  believe  that  this  reaction  is  of  im- 
portance, but  it  does  not  explain  all  the  facts  of  agglutination,  nor  is 
the  relation  between  agglutinating  and  precipitating  power  of  im- 
mune serums  a  constant  one.  In  support  of  this  hj^pothesis  is  the 
observation  of  Scheller^"  that  mixtures  of  typhoid  bacilli  and  agglu- 
tinating serum  lose  their  agglutinability  by  vigorous  shaking,  which 
may  be  interpreted  as  the  result  of  disintegration  of  the  agglutinating 
precipitate.  Shaking  of  either  bacteria  or  serum  alone  is  without' 
effect.  Neisser  and  Friedemann^^  found  that  if  the  bacterial  cells  were 
saturated  with  lead  acetate,  washed  in  water  until  all  soluble  lead  was 
removed,  and  then  treated  with  H2S,  they  were  promptly  agglutinated 
and  precipitated,  supporting  other  observations  that  indicate  that 
precipitation  within  the  bacterial  cells  can  lead  to  agglutination. 
This  sort  of  agglutination  is  related  to  the  process  of  formation  of 

"Brit.  Med.  Jour.,  June  14,  1919. 
"  Philippine  Jour.  Sci.,  1908  (3),  47. 
"Jour.  Path,  and  liact.,  1912  (17),  130. 
'8  Eisenberg  and  V^)lk,  Zeit.  f.  Infektionskr.,  1902  (40),  192. 
"^'8  For  comploto  bibliography,  sec  Craw,  Jour,  of  Hygiene,  1905  (5),  113. 
20  Cent.  f.  Bakt.,  1910  (54).  150. 
2'  Miinch.  nied.  Wocli.,  1904  (51).  4(15  and  S27. 


AGGLUTINATION  181 

coarse  floceuli  in  soJiitioiis,  and  probably  depends  uj)()n  alterations  in 
surface  tension. 

Bordet-^  made  the  important  observation  that  agglutination  does  \ 
not  occur  if  both  the  bacterial  suspension  and  the  agglutinating  serum 
are  dialyzed  free  from  salts  before^  mixing;  but  if,  to  such  mixtures,  a 
small  amount  of  NaCl  is  added,  agglutination  and  precipitation  of  the 
bacteria  occur  at  once.  This  observation  brought  the  phenomenon  of 
bacterial  agglutination  into  close  relation  with  the  precipitation  of 
colloids  by  electrolytes,  Bordet  comparing  it  to  the  precipitation  of 
particles  of  inorganic  matter  suspended  in  the  fresh  water  of  rivers 
that  occurs  when  the  fresh  water  meets  the  salt  water  of  the  ocean. 
He  found  that  the  agglutinin  combined  with  the  bacteria  in  the  ab- 
sence of  the  salts,  and  the  resulting  compound  was  precipitated  by  the 
addition  of  minute  amounts  of  electrolytes, ^'^  which  alone  did  not 
precipitate  or  agglutinate  the  bacteria  or  the  serum.  This  indicates 
that  the  agglutinins  cause  a  change  in  the  bacteria  which  brings  them, 
under  the  same  physical  laws  as  the  inorganic  colloidal  suspensions, 
which  are  characterized  by  being  precipitated  by  the  addition  of  traces 
of  electrolytes."'*  This  precipitation  is  undoubted!}^  due  to  changes 
in  solution  tension  and  surface  tension  (see  "Precipitation  of  Colloids, " 
introductory  chapter).  Before  the  agglutinin  combines  with  the 
bacteria  they  behave  like  the  colloidal  solutions  of  organic  colloids, 
being  precipitated  only  by  the  salts  of  heav}-  metals,  alcohol,  formalin, 
etc.,  or  by  great  concentrations  of  neutral  salts.  Field  and  Teague-^ 
have  found  that  agglutinins  carry  positive  charges  while  bacteria  are 
negative,  and  that  b}'  the  electric  current  agglutinins  can  be  separated 
from  bacteria  with  which  they  have  combined;  this  shows  that  the 
agglutinin  is  not  destroyed  in  the  reaction.  Teague  and  Buxton^^ 
consider  that  neutralization  of  the  electric  charge  of  the  bacteria  is  not, 
however,  the  only  important  factor  in  agglutination. 

According  to  Bechhold^^  normal  bacteria  behave  like  inorganic 
suspensions  that  have  each  particle  protected  by  an  albumin-like 
membrane,  which  prevents  them  from  being  thrown  out  of  suspension 
by  solutions  of  alkali  salts,  etc.  After  being  acted  on  by  agglutinin 
they  are  so  altered  that  they  behave  like  the  unprotected  inorganic 
suspensions,  and  are  precipitated  by  salts  and  other  electrolytes. 
This  suggests  the  possibility  that  the  agglutinin  makes  the  bacteria 

22  Ann.  d.  I'Inst.  Pasteur,  1899  (13),  225. 

23  Corroborated  for  sensitized  red  corpuscles  by  Eisner  and  Friedemann,  Zeit. 
Immunitat.,  1914  (21),  520. 

2*  Arrhenius  (Zeit.  physikal.  Chem.,  1903  (46),  415)  has  attempted  to  show 
that  the  gas  laws  are  applicable  to  the  partition  of  agglutinin  between  the  bacteria 
and  the  medium,  which  he  compares  to  the  partition  of  iodin  between  water  and 
carbon  disulphid.  This  idea  is  not  accepted  Vyy  Craw  {loc.  cit.),  nor  by  Drej'er 
and  Douglas,  Proc.  Roval  Soc,  1910  (82),  185. 

25  Jour.  Exper.  Med\,  1907  (9),  86. 

2«  Zeit.  phvsikal.  Chem.,  1907  (57),  76. 

2'  Zeit.  f.  physikal.  Chem.,  1904  (48),  385. 


182  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

permeable  for  these  electrolytes.  Buxton  and  Shaffer^**  also  found 
that  bacteria  which  have  been  acted  upon  by  agglutinin  behave  as 
if  their  proteins  had  been  so  changed  that  they  are  more  capable  of 
absorbing  or  combining  with  salts  than  when  in  their  normal  condition. 
Strong  salt  solutions  inhibit  agglutination  by  preventing  the  binding 
of  the  agglutinin.-^  Tulloch^"  observed  that  in  the  presence  of  salts 
of  mono-  and  di-valent  cations,  unsensitiz.ed  bacteria  do  not  readily 
precipitate  or  agglutinate,  but  sensitized  bacteria,  as  Bordet  showed, 
agglutinate  with  small  quantities  of  salts.  In  this  respect  unsensit- 
ized  bacteria  behave  like  "non-rigid  colloids, "  such  as  fresh  egg  white, 
while  sensitized  bacteria  resemble  "rigid  colloids,"  such  as  denatured 
egg  white.  Hence  he  advances  the  hypothesis  that  the  process  of 
sensitization  is  akin  to  that  of  denaturation  of  proteins,  the  specificity 
perhaps  depending  on  different  degrees  of  denaturation.  Mansfeld^^ 
would  bring  agglutination  into  line  with  other  serological  reactions  as 
a  protein  digestion  process,  by  his  hypothesis  that  bacteria  are  held 
in  suspension  by  protective  colloids  which  are  digested  by  an  enzyme, 
the  agglutinin.  He  finds  in  favor  of  this  hypothesis  that  the  tempera- 
ture and  reaction  curves  correspond  to  enzyme  actions,  that  agglu- 
tinating serum  contains  an  enzyme  digesting  protein  extracted  from 
bacteria,  and  that  during  agglutination  the  agglutinogen  is  destroyed. 

Agglutination  obeys  the  same  laws  as  other  similar  physical 
phenomena;  the  rate  of  agglutination  depends  upon  the  concentration 
of  the  suspension  and  of  the  electrolytes,  and  varies  with  the  valence 
of  the  cations.  Although  bacteria  in  an  electric  stream  move  toward 
the  anode  like  all  suspensions,  after  being  acted  on  by  agglutinin  they 
are  agglutinated  by  the  current  between  the  poles  ;^2  this  indicates 
the  importance  of  the  electrical  charges  of  the  bacterial  surfaces  in 
their  agglutination  reactions. 

In  all  respects  the  behavior  of  bacteria  and  agglutinin  resembles 
the  behavior  of  colloidal  mixtures  in  suspension  (Neisser  and  Friede- 
mann)'*''  which  form  an  electrically  amphoteric  colloidal  suspension, 
so  that  the  ions  of  electrolytes  or  the  electric  currents,  by  discharging 
them  unequally,  cause  precipitation.  Physico-chemical  researches, 
however,  have  yet  failed  to  explain  the  specific  character  of  the  ag- 
glutinins for  specific  bacteria,  but  Michaelis^'*  has  developed  an  inter- 
esting analogy  in  the  specific  agglutination  of  bacteria  by  acids.     This 

28  Zeit.  pliy.sikal.  Chem.,  1907  (57),  47. 

29  Landstoinor  and  Ht.  VVclocki,  Zoit.  Iiuiiiunitiit.,  1910  (8),  397. 
5"  Biocheni.  Jour.,  1914  (8),  293. 

3' Zeit.  Iiiununitut.,  1918  (27),  197. 

"  Bocliliold;  liowover,  liuxton  and  Teague  (Kolloid  Zcitschr.,  1908,  II,  Suppl. 
2)  8tat(!  tliat  agglutinin  bacteria  do  move  towards  tlie  anode,  but  slower  tli.'in 
normal  bat-teria. 

"  Miincli.  med.  Woch.,  1904  (51),  405  and  827;  see  also  ( uranl-Mangin  and 
llemi,  (.'onipt.  Rend.  Soc.  I'iol.,  1904,  vol.  5();  ami  Zangger,  Cent.  f.  Hakt.  (ref.), 
1905  (3()),  225. 

^*  Folia  Serologica,  1911  (7),  1010;  akso  IJeniasch,  Zeit.  Iiumunitat.,  1912  (12), 
208. 


AGGLUTINATION  183 

is  based  on  the  fact  that  the  optimum  concentration  of  H-ions  which 
precipitates  proteins  from  solution  is  characteristic  and  constant  for 
each  protein,  and  the  same  is  true  for  the  aRglutination  of  bacteria 
by  acids,  the  a^shitination  by  acids  beinp;  even  more  sharply  specific 
in  some  cases  than  the  agglutination  l)y  immune  sera;  e.  g.,  typhoid 
and  paratyphoid  bacilli  are  readily  distinguished  because  the  former 
are  agglutinated  by  a  concentration  of  H-ions  from  4  to8X10~^,  while 
paratyphoids  require  16  to  32X10"^,  and  colon  bacilli  are  not  agglu- 
tinated at  all  by  acids.  The  acid  agglutination,  however,  does  not  al- 
ways affect  all  strains  in  the  .same  way,  some  strains  which  are  not 
readily  agglutinable  by  antisera  also  resisting  acid  agglutination.^' 
According  to  Arkwright,^''  typhoid  bacilli  contain  two  extractable 
proteins  that  are  agglutinated  by  acids,  one  at  3.6X10"^  and  the  other 
at  1.1  X 10"^;  the  former  seems  to  be  related  to,  if  not  identical  with, 
the  substance  that  is  precipitated  by  immune  serum.  Apparently 
acid  agglutination  of  bacteria  belongs  to  the  same  class  of  reactions  as 
the  coagulation  by  H-ions  of  amphoteric  colloids  of  preponderatingly 
acid  character.  Bacteria  which  have  been  sensitized  by  serum  are 
more  sensitive  to  acid  agglutination  than  are  normal  bacteria." 

Alterations  in  the  agglutinability  of  bacteria  are  marked,  e.  g., 
strains  of  typhoid  bacilH  freshly  cultivated  from  human  infections 
may  be  practically  inagglutinable  even  by  active  serum,  but  after  pro- 
longed cultivation  on  media  they  may  or  may  not  develop  agglutina- 
bilit}^  This  phenomenon  has  not  yet  been  satisfactorily  explained, 
but  it  may  depend  on  an  active  immunity  of  the  bacteria  against  the 
agglutinins.  Such  bacteria  injected  into  rabbits  produce  antisera  that 
will  agglutinate  ordinary  agglutinable  strains,  but  not  themselves; 
hence  they  do  not  lack  agglutinogens.  They  give  normal  complement 
fixation  reactions,  and  hence  do  not  lack  receptors,  and  they  agglu- 
tinate with  acids  and  chemicals  much  the  same  as  ordinary  agglutin- 
able strains. ^^  Moreover,  identical  strains  of  bacteria  grown  on  media 
of  different  composition  may  show  considerable  variations  in  agglu- 
tinability (Dawson). ^^ 

Conglutination. — Under  this  term  Bordet  and  Gay  described  the  observation 
that  in  ox  serum  there  is  a  substance  wliich  combines  with  corpuscles  (or  bacteria) 
that  have  been  acted  upon  by  agglutinating  sera,  and  augments  the  agglutina- 
tion.'"' Dean  finds  that,  in  general,  agglutination  requires  two  agents,  one  being 
the  specific  antibody,  and  the  other  a  precipitable  substance,  probably  a  globulin. 
When  cells  have  combined  with  the  antibody  the  precipitable  substance  is  aggre- 
gated on  their  surfaces,  and,  presumably,  determines  the  agglutination.  Co-agglu- 
tination, described  by  Bordet  and  Gengou  as  the  agglutination  bj'  an  antigen  and 
the  homologous  antibody,  of  the  corpuscles  of  another  animal,  is  probabh-  closely 
related  to  these  phenomena  (Dean). 

"  See  Kemper,  Jour.  Inf.  Dis.,  1916  (18),  209. 
3«  Zeit.  Immunitat.,  1914  (22),  396;  Jour.  Hyg.,  1914  (14),  261. 
'Mvrumwiede  and  Pratt,  Zeit.  Immunitat.,  1913  (16),  517. 
=8  Mcintosh  and  McQueen,  Jour   Hyg.,  1914  (12),  409. 
"  Jour.  Bact.,  1919  (4),  133. 

"Literature  given  bv  Dean,  Proc.  Royal  Soc.  (B),  1911  (84),  416;  Hall,  Univ. 
CaUf.  Publ.,  Pathol.,  19i3  (2),  111. 


184  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

PRECIPITINS" 

If  to  a  solution  containing  proteins  we  add  in  proper  proportions 
the  serum  of  an  animal  immunized  against  the  same  protein,  a  pre- 
cipitate will  soon  form.  While  not  absolutely  specific,  the  quantitative 
specificity  of  the  precipitin  reaction  is  sufficiently  characteristic  to  be 
of  great  value  in  biological,  bacteriological,  and  medicolegal  work, 
and  it  is  of  importance  to  the  phj^siological  chemist,  since  it  furnishes 
a  means  of  distinguishing  between  closel}^  related  forms  of  proteins, 
more  delicate  by  far  than  any  known  chemical  reagent.  The  serum 
reactions  also  prove  that  there  are  sometimes  essential  differences  be- 
tween the  proteins  of , different  species  of  animals,  even  when  by  all 
other  methods  these  proteins  seem  to  be  practically  identical;  e.  g., 
lactalbumin  of  cow's  milk  is  in  some  respect  different  from  lactal- 
bumin  of  goat's  milk  since  it  produces  a  different  precipitin.  Medi- 
colegally  they  offer  an  accurate  method  of  determining  the  origin  of 
blood  and  serum  stains,  no  matter  how  old  the  stain  may  be;  thus 
Hansemann'*'-  found  that  material  obtained  from  a  mummy  5000 
years  old  gave  the  precipitin  reaction.'*^ 

Production  of  Precipitins. — For  the  production  of  the  precipi- 
tation reaction  it  is  necessary  to  have  in  the  substance  used  for  immu- 
nization a  certain  group,  the  precipitinogen,  which  when  injected  gives 
rise  to  production  of  precipitin  by  the  animal.  Apparonth'  almost 
any  protein  may  act  as  a  precipitinogen  if  injected  into  the  proper  ani- 
mal, but  it  must  he  a  foreign  protein;  rabbit  serum  will  not  pioduce 
precipitins  if  injected  into  a  rabbit, "*•*  probably  because  it  is  normally 
present  in  the,  blood  of  the  rabbit  and  therefore  does  not  stimulate 
any  reaction;  but  certain  chemical  alterations  in  the  proteins  of  an 
animal,  such  as  hegiting,  iodizing,  or  partial  digestion,  may  render  them 
so  different  from  the  normal  proteins  of  the  same  animal  that  they  will 
act  as  an  antigen  when  present  in  the  blood  of  that  animal,  or  another 
of  the  same  species,  from  which  they  were  derived.  Of  the  natural 
proteins  of  serum  the  globulins  arc  much  more  active  precipitinogens 
than  the  albumins.  In  general  the  more  foreign  the  protein,  the 
greater  the  amount  of  precipitin;  closely  related  animals,  e.  g.,  rabbit 
and  guinea-pig,  produce  little  precipitin  for  one  another's  proteins. 
This  indicates  distinctly  that  difference  in  species  depends  upon  orj  is 
associated  with  difference  in  chemical  composition  of  the  proteins. 
Different  species  of  animals  have  very  different  capacity  for  produc- 
ing precipitins,  rabbits  producing  active  sera,  while  guinea-pigs  can 

^'  For  complete  l)ibliogiaphy  of  the  suliject  of  "Precipitins"  see  the  r('>suin('  by 
Michaclis,  Ujjpenheinier's  llaiull).  il.  Hiochcinic.  I'.IOK,  II  (1),  bi>'2;  Kraus,  KoUc 
and  Wassiirinann's  llandb.,  1913,  II;  Llilenliiitli  and  Stel't'enliagen,  ibid.,  Ill,  257; 
Zinsser,  "Infection  and  Resistance." 

«  Munch,  nied.  Woch.,  1904  (30),  572. 

"  Not  corroborated  by  Schnudt,  Zeit.  allg.  Pliysiol..  1907  (7),  309. 

^*  Rarely  a  slight  reaction  against  homologous  i)roteins  has  been  obtained  {iso- 
precipitins). 


PRECIPITINS  185 

produce  hut  fcobl}'  precipitating  sera.  Cantacuzene''"  believes  that 
precipitins  are  formed  chiefly  in  the  lymphoid  tissues  and  bone  marrow, 
and  that  the  mononuclear  macrophages  are  most  active  in  their  for- 
mation." This  view  is  supported  by  the  observations  of  Hektoen,''^ 
that  anj''  agent  that  injures  the  bone  marrow  and  lymphoid  tissues 
(e.  (J.,  Roentgen  rays),  tends  to  interfere  with  antibody  production. 

Apparently  only  proteins  can  pioduee  precipitins;  when  split  to 
the  peptone  stage  they  lose  this  property,  but  the  proteins  of  serum 
resist  tryptic  digestion  a  long  time  before  losing  their  precipitinogcnic 
property,""*  which  is  destroyed  much  more  quickly  by  pepsin-HCl 
mixtures.  The  precipitate  itself  is  very  resistant  to  disintegrative 
agencies,  including  putrefaction  (Friedbergor),^^  but  is  soluble  in  dilute 
acids  and  alkalies.  It  has  the  power  of  binding  complement  (Gay)*'' 
and  if  the  complement  causes  solution  of  the  precipitate,  poisonous 
substances  are  formed  (Friedberger).  Excess  of  antigen  prevents  the 
formation  of  precipitate,  or  redissolves  it,  but  excess  of  antiserum  has 
no  effect.  Since  both  reacting  substances  are  colloids  they  follow  the 
laws  governing  other  mutually  precipitating  colloids,  and  precipitation 
occurs  only  when  they  are  brought  together  in  concentrations  that  lie 
within  definite  zones  of  relative  proportions.  It  is,  of  course,  perfectly 
possible  to  have  a  union  of  precipitin  and  antigen  without  any  visible 
))recipitate  occurring,  since  the  product  of  the  reaction  is  not  neces- 
saiily  insoluble  under  all  conditions;  in  this  case  the  occurrence  of  a 
reaction  must  be  demonstrated  by  some  other  method,  e.  g.,  the  com- 
plement fixation  reaction.  At  present  it  is  not  established  that  pre- 
cipitins can  be  secured  against  lipoids  or  other  non-protein  substances. 
Possibly  precipitins  can  be  produced  for  closely  related  substances  with 
molecules  approximating  in  size  the  protein  molecule,  e.  g.,  certain 
substances  present  in  supposedly  protein-free  filtrates  of  bacterial 
cultures.  As  with  the  agglutinin  reaction,  electrolytes  must  be 
present  or  precipitation  will  not  occur.  Neither  the  precipitin  nor  the 
antigen  seems  to  be  altered  appreciably  by  the  reaction,  since  when 
either  is  separated  from  the  precipitate  it  retains  its  original  properties. 

Since  precipitation  of  colloids  is  accompanied  by  or  dependent 
upon  an  aggregation  of  their  particles,  the  precipitin  reaction  is 
closely  related  to  the  agglutination  reaction.  The  amount  of  precip- 
itation obtained  is  much  modified  b}^  the  amount  of  inorganic  salts 
present,  and,  according  to  Friedemann,*^  there  is  a  general  resem- 

^5  Ann.  Inst.  Pasteur,  190S  (22),  54. 

*^  Spleen  tissue  cultivated  artificially  in  the  presence  of  horse  serum  produces 
specific  precipitins  for  horse  serum,  and  tissue  from  the  spleen  of  a  guinea  pig 
that  has  received  injections  of  horse  serum  also  develops  precipitins  for  horse 
serum  when  grown  in  cultures  (Prvzgode,  Wien.  klin.  Woch.,  1914  (27),  201). 

^^  Jour.  Infect.  Dis.,  1915  (17),  415;  1918  (22),  28. 

*^  Fleischmann,  Zcit.  klin.  Med.,  1906  (59),  515. 

"  Cent.  f.  Bakt.,  1907  (43),  490. 

50  See  Univ.  of  Calif.  Publ.  Pathol.,  1911  (2),  1. 

51  Arch.  f.  Hyg.,  1906  (55),  361. 


U 


186  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

blance  between  the  precipitin  reactions  and  the  precipitations  occur- 
ring when  colloids  precipitate  one  another;  i.  e.,  when  an  amphoteric 
colloid  reacts  with  either  an  acid  or  a  basic  colloid. ^^  So  far,  however, 
attempts  to  interpret  the  precipitin  reaction,  as  Arrhenius  has  tried 
to  do,  on  the  basis  of  the  laws  of  physical  chemistry,  have  not  met 
with  much  success  (Michaelis).  We  prefer  the  attitude  of  Krogh,^^ 
who  states  that  the  colloidal  chemical  part  of  immunological  reactions 
is  to  be  looked  upon  as  only  a  preliminary  step  to  the  real  chemical 
process  that  completes  the  reaction  and  gives  it  the  specific  characters. 
As  mentioned  in  the  preceding  section,  agglutination  of  bacteria  is  be- 
lieved to  be  independent  of  the  precipitins,  although  very  probably 
influenced  by  them.  As  wdth  all  the  other  substances  of  this  class,  the 
precipitins  have  a  haptophore  group  by  which  they  unite  to  the  protein 
molecule,  and  another  group  by  which  they  produce  the  change  re- 
sulting in  precipitation.  When  the  latter  group  is  destroyed  by 
heating  to  72°,  the  precipitin  is  converted  into  a  precipitoid,  which 
possesses  the  property  of  preventing  the  precipitation  of  unheated 
precipitin  by  the  specific  antigen.^'* 

The  immune  serum  contains  the  precipitin,  which  is  the  passive 
reagent  that  is  thrown  down  by  a  trace  of  the  immunizing  material 
(precipitinogen).  The  resulting  precipitate  is  the  insoluble  modifica- 
tion of  the  previously  dissolved  precipitin,  and  originates  chiefly  or 
entirely  in  the  proteins  of  the  immune  serum, ^'^  according  to  the  work 
of  Welsh  and  Chapman,  especially.  But  as  the  precipitate  is  able  to 
sensitize  anaphylactically,  both  actively  and  passively,  it  would  seem 
that  it  must  contain  both  the  antibody  (which  confers  passive  sensi- 
tization) and  antigen,  to  cause  active  sensitization  (Weil).^^  The 
precipitate  may,  when  of  maximum  amount,  contain  more  nitrogen 
than  corresponds  to  the  entire  euglobulin  of  the  immune  serum,  and 
the  euglobulin  contains  all  the  precipitin,  so  it  seems  probable  that  the 
precipitate  consists  of  more  than  the  precipitin  alone;  it  maj^  be  added 
that  the  precipitate  is  always  less  in  amount  than  the  total  globulin  of 
the  antiserum. ^^  It  is  always  greater  when  the  reaction  is  between 
homologous  antiserum  and  antigen,  than  with  even  closely  related  but 
heterologous  antigens,^**  so  that  the  quantitative  measurement  of  the 
amount  of  precipitate  is  of  value  in  applying  this  reaction  to  deter- 
mine the  nature  of  protein  solutions.  The  dilution  of  the  reacting 
solutions  is  of  influence,  however,  for  if  in  too  dilute  solutions  weak 

"  See  Friedemann  and  Friedenthal,  Zeit.  exp.  Path.  u.  Ther.,  190G  (3)  73; 
Iscovesco,  Compt.  Rend.  Soc.  Biol.,  1906,  Vol.  61,  and  subsequent  volumes. 

"  Jour.  Infect.  Dis.,  191G  (19),  452. 

^*  Precijntinogens  are  relatively  resistant  to  moderate  heating,  and  heated 
extracts  of  bacteria  are  used  for  precipitin  tests  under  tlie  name  thermoprccipiiiiis. 
See  review  by  A.  Ascoli,  Virchow's  Arcli.,  1913  (213),  182. 

"  Moll,  Zoit.,  exp.  Path.  u.  Ther  1900  (3),  325;  Welsh  and  Chapman,  Proc. 
Royal  Soc,  B.,  190<S  (SO),  Kil;  Zeit.  Immunitat.,  1911  (9),  517. 

"Jour.  Immunol.,  191()  (1),  35. 

"  Francosclielli,  Arch.  f.  Hvg.,  1907  (09),  207. 

"  Welsh  and  Chapman,  .Jour.  Hygiene,  1910  (10),  177. 


PRECIPITINS  187 

precipitins  may  fail  to  give  reactions;  with  strong  precipitins   the 
influence  of  dilution  is  much  less  (Michaelis). 

According  to  the  source  of  the  protein  used  we  recognize  bacterial 
"precipitins,  phyto-prccipitins  (for  plant  proteins), ^^  and  zooprecipi- 
tins  (for  animal  proteins).  Although  tissue  extracts,  body  fluids, 
and  exudates  are  generally  used  in  immunizing,  purified  constitu- 
ents of  these  protein  mixtures  will  also  excite  precipitin  formation, 
e.  g.,  we  may  immunize  with  caseinogen  as  well  as  with  milk.  Com- 
plete pepsin  digestion  of  proteins  deprives  them  both  of  their  precipi- 
tabihty  and  their  powder  to  produce  precipitins,  the  former  property 
being  lost  first.  Trypsin  seems  to  produce  the  same  effect  more  slowly. 
Some  of  the  fractions  of  protein  cleavage  may  be  slightly  precip- 
itinogenic  (Fink);''"  Heating  to  coagulation — indeed,  heating 
in  the  autoclave —  does  not  destroy  the  precipitinogenous  property  of 
proteins,  but  modifies  somewhat  the  reactions  of  the  precipitin  ob- 
tained," and  precipitinogen  is  destroyed  by  alkalies.  The  specificity 
of  precipitinogens  is  so  modified  by  heating  that  the  precipitins  en- 
gendered by  a  boiled  antigen  react  with  the  boiled  antigen  and  wuth 
similarly  heated  antigens  from  other  species,  but  not  w'ith  unheated 
antigens  even  from  the  homologous  species.''- 

As  proteins  introduced  into  the  stomach  are  normally  destroyed 
before  being  absorbed,  they  do  not  enter  the  blood  and  cause  pre- 
cipitin formation.  However,  as  is  well  known,  eating  of  excessive 
amounts  of  egg-albumen  or  other  easily  absorbed  proteins  may  re- 
sult in  theii'  passing  the  barriers  and  entering  the  blood  stream,  and 
in  this  way  precipitins  have  been  experimentally  produced.  Pre- 
sumably the  precipitin  reaction  is  a  means  of  throwing  such  foreign 
proteins  out  of  solution  and  rendering  them  harmless.  According 
to  Zinsser''^  and  others,  the  function  of  the  precipitin  is  to  sensitize 
the  unformed  foreign  proteins  to  the  digestive  complement,  a  view 
in  harmony  with  the  prevailing  tendency  to  correlate  the  immunity 
reaction  with  defense  through  enzymatic  hydrolysis. 

Precipitin  appears  in  the  blood  generall}'  about  six  days  after  in- 
jection of  the  protein,  but  disappears  after  injection  of  each  subse- 
quent dose  of  protein,  to  reappear  again  after  a  somewhat  shorter 
lapse^of  time.  After  injections  are  stopped,  the  precipitin  disap- 
pears rather  rapidly,  but  never  appears  in  the  urine,  although  it 

°^  Literature  on  precipitins  for  vegetable  proteins  given  by  Wells  and  Osborne, 
Jour.  Infect.  Dis.,  1911  (8),  66. 

«"  .Jour.  Infect.  Dis.,  1919  (25),  97.       _ 

^'  See  Obermayer  and  Pick,  who  consider  in  detail  the  effects  of  various  modi- 
fications of  proteins  upon  their  power  to  incite  precipitin  formation  (Wien.  klin. 
Woch.,  1900  (19),  327);  also  Landsteiner  and  Lampl,  Biochem.  Zeit.,  1918  (86),  343. 
The  precipitability  of  the  serum,  or  its  power  to  produce  precipitins,  is  not  affected 
by  disease  (Pribram,  Zeit.  exp.  Path.  u.  Then,  1906  (3),  28). 

«2  Schmidt,  Biochem.  Zeit.,  1908  (14),  294;  1910  (24),  45;  Zeit.  Immunitat., 
1912  (13),  173;  also  Zinsser,  "Infection  and  Resistance,"  1914,  p.  260. 

"  Jour.  Exper.  Med.,  1912  (15),  529;  1913  (IS),  219. 


188  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

may  enter  the  fetal  blood  from  the  blood  of  pregnant  female  animals. 
The  presence  of  precipitins  in  the  blood  does  not  seem  to  prevent 
the  excretion  of  the  foreign  protein  in  the  urine,  nor  are  the  animals 
less  susceptible  to  the  toxic  action  of  the  foreign  protein;  indeed,  the 
reaction  is  even  stronger  in  the  immunized  animals,  and  sometimes 
the  ordinary  dose  becomes  fatal.  Precipitin  and  antigen  may  coexist 
ununited  in  the  circulating  blood  under  certain  conditions. "^^  Certain 
antibodies  are  carried  down  with  the  precipitates  formed  when  the 
serum  containing  them  reacts  under  proper  conditions  with  an  anti- 
serum; e.  g.,  diphtheria  antitoxin  is  precipitated  when  added  to  the 
serum  of  a  rabbit  immunized  to  horse  serum.  This  is  not  true  of  all 
antibodies,  however, '^^  As  the  precipitates  formed  in  the  precipitin 
reaction,  when  injected  into  a  guinea-pig  make  it  passively  hj'persensi- 
tive  to  the  protein  used  as  antigen  in  the  precipitin  reaction,  it  would 
seem  that  the  precipitin  and  the  anaphylactin  are  identical  (Weil),®^ 
or  at  least  closely  associated. 

Chemical  Properties. — In  its  chemical  nature  precipitin  reseml^les  the  "anti- 
bodies" generallj',  being  precipitated  in  the  euglobulin  fraction  of  the  serum, ^' 
and  slowly  destroyed  by  trypsin,  rapidly  by  pepsin.  It  cannot  be  separated 
from  the  serum  proteins.  The  precipitation  by  precipitins  is  not  an  enzjmie 
action,  for  the  precipitins  are  used  up  in  the  process.  It  apparently'  does  not 
differ  from  precipitations  of  colloids  by  other  colloids  of  opposite  electrical  charges, 
except  in  that  the  reaction  is  specific. 

OPSONINS^s 

The  correlation  of  phagocytic  and  serum  immunity  was  accomplished  when 
A.  E.  Wright  showed  that,  before  any  considcrat)le  phagocA^tosis  of  bacteria 
can  take  place,  the  bacteria  must  first  be  acted  upon  by  serum,  which  in  some  way 
prepares  them  to  be  ingested  by  the  leucocj^tes.  The  hj-pothetical  substances 
accomplishing  this  sensitization  of  the  bacteria  were  called  opsonins  by  Wright 
and  they  exist  to  a  certain  extent  in  normal  serum,  being  increased  bj-  immuniza- 
tion. Not  only  bacteria,  but  cellular  elements  in  general,  including  especially 
red  corpuscles,  and  even  unorganized  particles  (such  as  melanin), "^^  are  sensitized 
for  phagocytosis  by  opsonins.  Probably  phagocytosis  by  endothelial""  and  other 
cells  also  requires  sensitization  of  the  bacteria  by  opsonins.  Although  there  have 
been  many  expressions  of  the  opinion  that  the  opsonins  are  not  distinct  antibodies, 
but  are  identical  with  agglutinins,  bacteriolytic  amboceptors,  or  other  antibodies, 
there  is  much  evidence  to  the  contrary."'  However,  the  union  of  opsonin  and 
bacteria  seems  to  follow  the  same  quantitative  laws  as  otlior  antigen-antibody 
reactions  (Amato)." 

There  are  two  opsonizing  elements  in  serum,  one  thermostable  and  one  thermola- 
bile,  it  being  the  former  which  is  increased  during  immunization;  the  thermostable 

"  Bayne-Jones,  Jour.  Exp.  Med.,  1917  (25),  837. 

^^  8ee  Gay  and  Stone,  Jour.  Immunol.,  191(5  (1),  83. 

"  Jour.  Immunol.,  1910  (1),  1. 

6'  Funck  (Cent.  f.  Bakt.  (Kef.),  1905  (30),  744)  states  that  if  the  precipitin 
serum  is  very  strong,  part  of  the  precipitin  conies  down  in  the  pscudoglobulin. 

**  Bibliography  given  by  Neuield,  Kolle  and  Wasscrmann's  Handhucii,  1913 
(2),  440. 

«»  Shattock  and  Dudgeon,  Proc.  Royal  Soc.  (B),  1908  (80),  165. 

70  Briscoe,  .lour.  Patli.  and  Bact.,  1907  (12),  GO.  iSee  also  Manwaring  and  Coe, 
who  found  tliat  tlie  Kupffer  cells  can  take  up  only  opsonized  pneumococci  (Proc. 
See.  Exp.  Biol.,  191G  (13),  171). 

"  See  Ilcktoen,  Jour.  Infec.  Dis.,  1909  (0),  78;  1913  (12),  1. 

"  Sporimentale,  1918  (71),  459. 


OPSONINS  189 

clement  unites  firmly  with  the  object  which  is  to  l)o  opsonized,  while  the  thermo- 
labile  clement  seems  to  remain  free  in  the  fluid  (ITektocn).^' 

It  would  seem  that  opsonization  and  phagocytosis  constitute  but  one  of  a  series 
of  similar  processes  by  which  foroif:;n  proteins  are  removed  from  the  blood  and  tis- 
sues; i.  e.,  by  lysis  by  extracellular  enzymes  when  this  is  possible,  as  it  is  with 
simple  protein  aggregates  (albuminolysis  )and  with  some  of  the  more  labile  cells 
Oiemolj'sis,  bacteriolysis);  but  in  the  case  of  more  resistant  structures,  notably 
Gram-positive  cocci  and  acid-fast  bacilli,  extracelhilar  lysis  being  unsuccessful, 
these  protein  structures  are  taken  within  the  cells  where  a  greater  concentration  of 
enzymes  may  destroj"-  them.  Fundinncntnlly  serum  hndcriolysis  and  phagocytosis 
seem  to  he  the  same — in  each  case  specific  antibody  sensitization  prepares  the  bacterium 
for  lysis  by  enzymes,' either  inside  or  outside  the  cells  that  furnish  the  lytic  enzyme. 

As  yet  nothing  is  known  concerning  the  change  brought  about  in  the  bacteria 
by  the  opsonin,  although  it  has  been  established  that  it  is  the  bacteria  that  are 
modified  and  not  the  leucocytes.  The  chemical  nature  of  the  opsonins  is  likewise 
unknown,  except  that  they  may  combine  with  certain  inorganic  ions  and  are  then 
inert  (Hektoen  and  Ruediger),'''  since  addition  of  CaCU,  BaClj,  SrCl2,  MgCU, 
KoSO^,  NallCOs,  sodium  oxalate  and  potassium  ferrocyanide,  inhibit  the  opsonic 
efTect  of  serum.  On  the  contrary,  calcium  salts  stimidatc  the  phagocytic  effect  of 
leucocytes,  salts  of  barium  and  strontium  being  inactive."*  In  common  with  other 
immune  bodies,  opsonins  are  thrown  down  in  the  soluble  serum  globulins.'^ 
They  are  very  sensitive  to  acids  and  alkalies,  being  destroyed  by  a  concentration 
of  n/i  and  their  maximum  effect  is  at  the  neutral  point."  However,  treatment  of 
either  the  bacteria  or  the  leucocytes  with  very  weak  acids  or  alkalies,  increases 
the  rate  and  amount  of  phagocytosis  (Oker-Blum)."'  Opsonins  may  be  developed 
by  immunizing  against  substances  practically  tree  from  protein,  c.  g.,  melanin 
granules."^  Injection  of  nu  'lein  preparations  may  in<'rease  the  amount  of  opsonin 
present  in  the  blood. ^°  Cholesterol  in  excess  dimini.shes  phagocytosis,  but  appar- 
ently through  its  action  on  the  leucocytes.*'  Both  the  sensitization  of  bacteria 
and  their  ingestion  by  leuco-^j^tes,  either  with  or  without  sensitization,  tajj^e  place 
in  accordance  with  the  laws  regulating  an  adsorption  process  (LediilEham,*^ 
S^-hutze'8).  ^. 

THE  MEIOSTAGMIN  REACTION 

Reav'tion  of  antigens  with  their  specific  antibodies  results  in  lowering  the  sur- 
face tension  of  the  solution  in  which  the  reaction  occurs,  which  may  be  demonstra- 
ted by  counting  the  number  of  drops  of  the  fluid  per  minute,  under  constant 
conditions.  Ascoli  and  Izar*'  worked  out  methods  for  practical  application  of  this 
phenomenon,  gi\ang  it  the  name  of  "meiostagmin  reaction,"  from  the  Greek, 
meaning  "small  drop."  The  number  of  drops  from  a  stalagmometer  is  counted, 
and  an  increase  of  two  or  more  per  minute  is  considered  a  positive  reaction,  after 
two  hours'  incubation  of  the  reacting  mixture;  the  in.'rease  is  seldom  above  eight 
drops.  This  reaction  is  said  to  be  sharply  specific  and  extremely  delicate,  detect- 
ing antigens  diluted  up  to  1  in  100,000,000  or  more.     The  antigens  used  are  soluble 

"Sawtchenko  (Arch.  Sci.  Biol.,  1910  (15),  145;  1911  (16),  161)  holds  that 
there  are  two  steps  in  phagocytosis:  (1)  Fixation  of  the  bacteria  to  the  leuco- 
CA'te  because  of  modification  of  surface  tension  by  the  fixative  substance  (opsonin 
or  amboceptor-complement  complex);  (2)  Ameboid  motion  of  the  phagocyte; 
an  entireh^  independent  phenomenon.  Neither  phase  of  phagoc3'tosis  can  occur 
in  the  absence  of  electrolytes. 

^*  Jour.  Infect.  Dis.,  1905  v2),  129. 

l^  Hamburger,  Biochem.  Zeit.,  1910  (24),  470;  1910  (26),  66. 

"^  See  Simon  et  al.,  Jour.  Exp.  Med.,  1906  (8),  651;  Heinemann  and  Gatewood, 
Jour.  Infec.  Dis.,  1912  (10),  416. 

""  Xoguchi,  Jour.  Exp.  Med.,  1907  (9),  454. 

^8  Zeit.  Immunitat.,  1912  (14),  485;     Schutze,  Jour.  Hvg.,  1914  (14),  201. 

'^Ledingham,  Zeit.  Immunitat.,  1909  (3).  119. 

80  Bedson,  Jour.  Path,  and  Bact.,  1914  (19),  19.. 

81  Dewev  andNuzum,  Jour.  Infect.  Dis.,  1914  (15),  472. 

82  Jour.  Hvg.,  1912  (12),  320. 

83Munch.  med.Woch.,  1910  (57),  62,  182  and  403. 


190  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

in  alcohol  but  their  nature  is  unknown;  the  antibody  involved  in  the  reaction  is 
referred  to  as  the  meiostagmin,  but  its  relation  to  other  antibodies  is  likewise 
unknown. 

THE  EPIPHANIN  REACTION 

Besides  reduction  in  surface  tension,  other  physico-chemical  changes  result 
from  antigen-antibody  reactions,  including  the  rate  of  diffusion,  the  osmotic 
pressure,  and,  in  consequence,  according  to  Weichardt,  the  neutral  point  to  phenol- 
phthalein  of  a  mixture  of  barium  hydroxide  and  sulphuric  acid,  is  also  changed 
towards  the  acid  side  by  antigen-antibody  reactions  taking  place  in  the  mixture.** 
This  phenomenon  has  been  utilized  by  Weichardt,  under  the  name  of  "epiphanin 
reaction,"  to  determine  the  occurrence  of  such  interaction  of  antigen 'and  antibody. 
The  reaction  probably  depends  upon  absorption  phenomena,  but  the  exact  nature 
of  the  change  is  not  \et  understood.  According  to  Rosenthal,*^  the  epiphanin 
reaction  is  especiall}'  suitable  for  demonstrating  cancer  antibodies  and  antigens, 
but  Burmeister*^  and  others  have  not  been  successful  with  this  procedure. 

8*  See  Weichardt,  Berl.  klin.  Woch.,  1911  (48),  1935;  Rosenthal,  Zeit.  Im- 
munitiit,  1912  (13),  383;  Angerer  and  Stott^r,  Mtinch  med.  Woch.,  1912  (59), 
2035. 

85  Zeit.  Chemotherapie,  1912  (1),  156. 

8«Jour.  Infec.  Dis.,  1913  (12).  459. 


CHAPTER  VIII 

CHEMISTRY    OF    THE    IMMUNITY    REACTIONS    (Continued). 
ANAPHYLAXIS     OR    ALLERGY,     ABDERHALDEN     REACTION. 

ANAPHYLAXIS  OR  ALLERGY 

In  many  instances  the  injection  of  a  foreign  protein  into  an  ani- 
mal produces  severe,  perhaps  fatal,  intoxication.  With  some  pro- 
teins this  natural  toxicity  is  very  marked, — thus  eel  serum  is  fatal 
to  rabbits  and  dogs  in  doses  of  0.1  to  0.3  c.c.  per  kilo,  and  foreign 
sera  are  commonly  toxic  to  other  animals;  e.  g.,  fresh  bovine  and 
human  serum  are  quite  toxic  to  guinea-pigs.  This  so-called  ''pri^ 
mary"  toxicity  is  reduced  or  destroyed  in  most  cases  by  heating  to 
56°  for  30  minutes.^  Almost  any  non-toxic  soluble  protein,  however, 
may  be  made  toxic  for  animals  by  giving  the  animal  a  small  dose  of 
this  same  protein  at  least  eight  days  previously.  This  preliminary 
dose,  which  may  be  extremely  minute, ^  renders  the  animal  hypersen- 
sitive to  the  same  protein,  so  that  a  relatively  small  quantity  (a  few 
milligrams  in  the  case  of  the  guinea-pig)  of  an  otherv.-ise  entirely 
harmless  protein,  such  as  eg^  white  or  milk,  produces  violent,  often 
fatal,  symptoms  when  introduced  into  the  blood  of  the  animal.  We 
shall  not  discuss  the  general  features  of  the  reaction,  its  history  and 
its  relation  to  biology  and  pathology,  which  are  fully  covered  in  many 
easil}^  accessible  reviews/''  but  shall  limit  our  consideration  to  the  more 
definitely  chemical  aspects  of  the  reaction.^ 

The  Substances  Involved  (Anaphylactogens). — As  far  as  now 
known,  these  are  always  proteins,  and  with  the  exception  of  gelatin^ 

^  The  nature  of  the  toxic  agent  is  unknown,  but  there  is  reason  to  believe  that 
it  is  formed,  at  least  in  part,  during  the  coagulation  of  the  drawn  blood. 

-  Julian  Lewis  has  found  that  if  a  very  small  amount  of  protein  is  injected  at 
the  same  time  as  a  large  dose  of  another  foreign  protein,  no  sensitization  results 
to  the  former;  presumably  the  available  cell  receptors  are  occupied  by  the  protein 
given  in  larger  amounts.     (Jour.  Infect.  Dia.,  1915  (17),  241.) 

^  Doerr,  Kolle  and  Wa.ssermann's  Handbuch,  1913,  Vol.  II;  and  Zeit.  f.  Im- 
munitat.,  1910;  (2,  ref.),  49;  also  v.  Pirquct,  Arch.  Int.  Med.,  1911  (7),  259; 
Friedmann,  Jahresber.  Ergeb.  Immunitatfrsch.,  1911  (6),  31;  Schittenhelm,  ibid., 
p.  115;  Hektoen,  Jour.  Amer.  Med.  Assoc,  1912  (.58),  1081;  Zinsser,  Arch.  Int. 
Med.,  1915  (16),  223.  Concerning  anaphylaxis  in  man  see  Longcope,  Amer.  Jour. 
Med.  Sci.,  1916  (152),  625.  Concerning  cutaneous  reactions  see  Kolmer,  Bull. 
Johns  Hopkins  Hosp.,  1917  (28),  163. 

■•  Many  of  the  chemical  features  of  anaphylaxis  I  have  covered  in  the  following 
series  of  articles:  Jour.  Inf.  Dis.,  1908  (5),  449;  1909  (6),  506;  1911  (8),  66;  1911 
(9),    147;  1913  (12),  341;  1914  (14),  364  and  377;  1915  (17).  259;  1916  (19),  183. 

*  Wells,  Jour.  Amer.  Med.  Assoc,  1908  (50),  527;  Jour.  Infect.  Dis.,  1908  (5), 
459;  Starin,  Jour.  Infect.  Dis.,  1918  (23),  139. 

191 


192  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

and  a  few  others,  practically  any  soluble  protein  will  produce  sen- 
sitization and  intoxication  of  susceptible  animals,  i.  e.,  almost  any 
soluble  protein  may  be  an  anaphylactogen.  As  with  the  other  immun- 
ity reactions,  observations  have  been  made  which  are  interpreted  as 
indicating  that  non-protein  substances  can  produce  this  reaction,  but 
these  interpretations  are  not  generally  accepted.  (See  Antigens,  Chap, 
vii.)  It  is  possible,  however,  for  non-protein  substances  to  combine 
with  or  alter  the  proteins  of  an  animal  so  that  they  become  as  foreign 
proteins  to  that  animal,  and  thus  cause  sensitization;  in  this  way  can 
be  explained  apparent  anaphylactic  reactions  to  iodin  and  arsenic  com- 
pounds and  other  non-protein  substances.''  As  far  as  my  own  experi- 
ments show,  nothing  less  than  an  entire  protein  molecule  will  suffice, 
the  products  of  protein  cleavage  all  being  inactive.^  Zunz^  and  Fink,^, 
however,  report  some  positive  results  with  proteoses.  Presumably  the 
inefficiency  of  gelatin  as  an  anaphylactogen  depends  upon  its  de- 
ficiency in  aromatic  radicals,  since  these  radicals  have  been  shown 
(Vaughan,  Obermeyer  and  Pick)  to  be  particularly  important  in 
immunological  reactions.  It  is  not  necessary  for  a  protein  to  con- 
tain all  the  known  amino-acids  of  proteins  to  be  active,  however,  for 
certain  vegetable  proteins  (zein,  hordein,  gliadin)  which  lack  one  or 
more  of  such  amino-acids  as  glycine,  tryptophane,  or  lysine,  pro- 
duce typical  reactions.  Some  compound  proteins  arc  efficient  ana- 
phylactogens  ( mucin, ^^  casein)  but  with  alpha-nucleoproteins  which 
have  been  thoroughly  purified  I  have  obtained  only  negative  results;" 
as  also  with  histon  and  nucleic  acid,  the  isolated  components  of  nu- 
cleins.  Globin,  from  hemoglobin,  is  also  non-antigenic.  Bacterial 
substances,  extracts  of  plant  tissues,  purified  plant  proteins,  and  pro- 
teins obtained  from  invertebrates  and  cold-blooded  vertebrates,  have 
all  been  found  to  be  anaphylactogens,  if  the}'  can  be  iqt reduced  bj^ 
any  means  into  the  blood  or  tissues  in  a  soluble  unaltered  condition. 

If  the  proteins  are  rendered  insoluble  bj^  coagulation  they  become 
inert,  but  proteins  which  cannot  be  made  insoluble  bj^  heating  [e.  g., 
casein,  ovomucoid)  withstand  boiling  temperatures.  Trypsin  de- 
stroys anaphylactogens  in  just  the  same  proportion  as  it  splits  the  protein 

«  See  Bottner  (Deut.  Arch.  klin.  Med.,  1918  (125),  1),  concerning  collargol 
anaphylaxis. 

^  Abderhaldcn  (Zeit.  physiol.  Chcin.,  1912  (81),  314)  states  that  he  has  ob- 
tained a  positive  reaction  with  a  synthetic  polypeptid  containing  14  amino-acid 
molecules,  including  only  leucine  and  glycocoll.  E.  Zunz  (Jour,  physiol.  patli. 
gen.,  1917  (17),  449)  reports  obtaining  positive  results  with  much  simpler  polypop- 
tids  (.■^-5  glycylglycine).  These  reactions  consisted  in  changes  in  blood  pressure 
and  coagulability  in  rabbits,  and  we  do  not  know  whether  tj'pical  shock  can  be 
obtained  in  guinea  i)igs  with  these  jxij^tids. 

^7Ainz  (ZciL.  liniuunit:it.,  1913  ((iO),  580). 

"Jour.  Infect.  J)is.,  1919  (25),  97. 

'"Elliott,  Jour.  Infect.  J)is.,  1914  (15),  501. 

"  See  review  in  Zeit.  Irninuniti'lt.,  1913  (19),  599,  conccriiiiig  alpha-nucloopro- 
tcins,  which  is  the  type  usually  designated  as  "  nucleoproteins."  I  have  found 
beta-nucleoprotcins  to  be  more  effective  antigens  (Jour,  liiol.  Chem.,  191G  (28), 


ANAPHYLAXIS  OR  ALLERGY  193 

molecules;  thus,  globulins  resist  trypsin  longer  than  albumins,  both 
as  regards  coagulability  and  anaphylactic;  activit}'.  Acids,  alkalies 
and  other  chemical  agents  may  modify  the  reactivity  of  proteins  in 
proportion  to  the  changes  in  solubility  or  constitution  which  they 
produce. '2 

The  amounts  of  protein  necessary  to  produce  reactions  in  guinea- 
pigs  are  very  small.  With  crystallized  egg  albumin  sensitivity  has 
been  produced  with  one  twenty-millionth  of  a  gram  (0.000,000,05 
gm.)  and  fatal  reactions  are  obtained  after  sensitization  with  one- 
millionth  of  a  gram.  No  other  animal  seems  to  be  so  sensitive  to  this 
reaction  as  the  guinea-pig,  however,  and  rabbits  and  dogs  require 
larger,  and  in  many  instances,  repeated  doses  to  render  them  ana- 
phylactic. Within  certain  limits  large  doses  are  less  effective  in  sen- 
sitizing guinea-pigs  than  small,  e.  g.,  one  milligram  of  most  proteins 
will  usually  be  much  more  effective  than  one  hundred  milligrams. 
White  and  Averj'^^  found  that  there  is  a  certain  relation  between  the 
minimum  sensitizing  and  the  minimum  intoxicating  dose;  with  ex- 
tremely minute  sensitizing  doses  a  larger  intoxicating  dose  is  required 
to  produce  fatal  reaction  than  when  the  sensitizing  dose  is  larger.  If 
too  large  intoxicating  doses  are  used,  however,  the  degree  of  reaction 
may  be  lowered. ^^ 

It  is  now  generally  assumed  that  both  the  sensitizing  and  intox- 
icating agent  are  (or  are  derived  from)  one  and  the  same  protein, 
but  the  minimum  intoxicating  dose  is  always  larger  than  the  mini- 
mum sensitizing  dose;  thus,  with  pure  egg  albumin  the  minimum 
lethal  dose  for  sensitized  pigs  was  one-twentieth  to  one-tenth  milli- 
gram by  intravascular  injection,  or  about  one  hundred  times  more 
than  the  minimum  fatal  sensitizing  dose.  With  less  soluble  proteins 
the  disparity  is  even  greater,  for  with  such  the  sensitizing  dose  is  not 
much  changed,  but  the  minimum  intoxicating  dose  is  relatively  much 
increased.  Apparently  an  animal  may  be  killed  by  much  less  antigen 
than  is  required  to  saturate  the  antibodies  present  in  its  body  (Weil). 
The  exact  fate  of  the  injected  antigens  is  unknown.  Manwaring^^ 
observed  no  loss  in  antigen  perfused  through  organs  of  sensitized 
animals,  but  others  have  found  that  antigen  injected  subcutaneously 
disappears  more  rapidly  in  sensitized  or  immunized  than  in  normal 
animals.'^ 

The  proteins  concerned  must  be  foreign  to  the  circulating  blood  of 
the  injected  animal,  but  they  may  be  tissue  proteins  of  the  same  ani- 
mal (e.  g.,  placenta  elements,  organ  extracts,  lens  proteins)  which 
are  not  normally  present  in  its  blood.  Indeed  it  has  been  claimed 
that  by  injecting  a  guinea-pig  with  the  dissolved  lens  of  one  eye  it 

12  See  Dold  and  Aoki,  Cent.  f.  Bakt.,  Ref.  Beilage  1912  (54),  246. 
"Jour.  Infect.  Dis.,  1913  riS),  103. 

1*  Terry  and  Andrews,  Proc.  Soc.  Exp.  Biol.  Med.,  1915  (12),  176. 
15  Jour.  Immunol.,  1917  (2),  511. 

i«  G.  H.  Smith  and  Cook,  Jour.  Immunol.,  1917  (2),  269. 
13 


194  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

will  become  sensitized  so  that  it  will  react  to  a  subsequent  injection 
of  the  lens  from  the  other  eye.^^  In  general,  tissue  proteins  are  less 
active  antigens  than  the  proteins  of  the  blood,  lymph,  and  secretions, 
but  even  keratins  maj^  produce  anaphylaxis  when  dissolved^^  and 
positive  results  have  been  obtained  with  proteins  from  mummies. ^^ 

The  Poisonous  Agent  (Anaphylatoxin). — The  sj^mptomatology  of 
the  intoxication  which  follows  injection  of  the  protein  into  an  animal 
sensitized  with  the  same  protein,  is  such  as  to  indicate  that  a  poison  is 
responsible,  although  as  yet  the  poison  has  not  been  isolated.  As  the 
symptom  complex  is  practically  the  same  no  matter  what  sort  of  protein 
is  being  used,  it  would  seem  that  the  poison  must  always  be  the  same 
or  similar — no  matter  how  varied  the  nature  of  the  proteins  capable  of 
inciting  anaphylactic  intoxication.  Probably  the  poison  is  a  product  of 
cleavage  of  protein  by  tissue  or  blood  enzymes,  which  act  only  in  the 
presence  of  the  specific  antibodies  which  unite  the  protein  to  the 
enzyme  (or  complement).  Vaughan  and  his  collaborators  showed  that 
proteins  boiled  with  an  alcoholic  NaOH  solution  might  be  split  into 
two  fractions,  one  toxic  and  alcohol-soluble,  the  other  non-toxic  and 
insoluble  in  alcohol.  The  toxic  fraction  gives  all  the  protein  reactions 
(except  that  of  Molisch  for  carbohydrates)  and  in  doses  of  8  to  100  mg. 
kills  guinea-pigs  with  symptoms  practically  identical  with  those  of 
anaphylactic  intoxication.  The  uniformity  of  the  toxic  effects  with 
preparations  from  different  sorts  of  proteins  suggests  the  existence  in 
every  protein  molecule  of  some  fundamental  toxic  group,  common  to 
all  proteins,  the  specificity  residing  in  other  non-toxic  attached  groups. 
This  and  other  observations  led  him  to  the  hypothesis  that  specific 
enzymes  are  developed  in  response  to  the  presence  of  foreign  pro- 
teins in  the  blood  stream,  and  that  upon  injection  of  a  second  dose 
of  the  same  protein  these  enzymes  at  once  disintegrate  it,  and  some 
of  the  cleavage  products  being  toxic  the  anaphylactic  intoxication 
results.  Many  of  the  later  developments  in  this  field,  especially 
Abderhalden's  studies  on  "protective  ferments,''  have  added  support 
to  this  hypothesis,  so  that  in  its  fundamental  conceptions  it  is  now  the 
most  generally  favored  explanation  of  the  processes  involved  in  ana- 
phylaxis. ^^ 

Friedberger  carried  the  matter  a  step  farther  by  showing  that  if 
serum  from  a  sensitized  animal  is  incubated  for  a  short  time  with  the 
same  protein,  and  in  the  presence  of  enough  complement,a  poison 
is  developed  which  produces  the  typical  symptoms  of  anaphylactic 
intoxication  when  injected  into  guinea-pigs.  This  poison  resists  heat- 
ing at  56°,  but  not  at  65°,  and  is  not  a  true  toxin,  for  it  will  not  jiroduce 

»'  Uhlenhutli  and  IliioiRld,  Zoit.  f.  Iiuinunitat.,  1910  (4),  7l)l. 

'8Krusiii«,  Arch.  f.  Aiifr<'nlioilk.,  Sup])!.,  11)10  (47),  47;  Clough,  Arb.  kais; 
Gesundlitsamic,  1911  Oil),   131. 

'»  llil.'iihulli,  Zcil.  f.  Imiininitat.,  1910  (4),  774. 

"  See  VauglKUi,  Aiiier.  Jour.  xMed.  Sei.,  19i;{  (145),  101;  Zeit.  Imiiuinitat.,  1911 
(9),  458.     ALso  a  full  review  in  his  "Protein  Split   Products,"  Philadeii)hia,  1913. 


.lAM/'//17..lA7^'  OH  ALLERiiY  195 

an  antitoxic  iniiuunity.  In  the  absence  of  complement,  or  when  the 
complement  fixation  is  prevented  by  strong  salt  solution, ^^  the  poison 
(anaplu'latoxin)  does  not  develop,  so  that  the  anaphylactic  reaction 
falls  into  the  same  class  as  the  lytic  reactions,  in  which  the  non-specific 
serum  complement  is  united  to  a  cell  by  the  specific  amboceptor,  and 
then  causes  lysis  of  the  cell;  in  anaphylaxis  not  an  organized  cell  but 
a  comi^lex  protein  molecule  is  disintegrated  by  the  complement,  but 
in  either  case  a  poisonous  substance  may  be  liberated. 

This  agrees  with  Vaughan's  hypothesis  in  ascribing  the  poisoning 
to  products  of  protein  disintegration  formed  by  enzyme  action,  but 
differs  in  that  specific  intermediary  substances  or  amboceptors  are  sup- 
posed to  be  developed  by  sensitization,  rather  than  specific  enzymes. 
Friedberger  is  of  the  opinion  that  many  or  all  the  different  immunity 
reactions  depend  upon  a  single  antibod}',  the  different  reactions  merely 
being  different  methods  of  demonstrating  the  presence  of  the  antibody 
in  the  serum.  The  precipitin  reaction  differs  from  the  anaphylactic 
reaction,  he  contends,  only  in  that  in  the  latter  the  specific  pre- 
cipitate is  dissolved  by  complement,  yielding  the  anaphylatoxin. 
There  are  many  objections'^  to  accepting  this  idea  in  its  entirety 
which  we  shall  discuss  later,  but  the  formation  of  a  poison  resembling 
that  of  anaphylaxis,  by  a  digestive  action  of  complement  fixed  to  the 
antigen  by  the  antibody,  seems  to  be  well  established,  both  as  regards 
in  vitro  and  in  vivo  reactions. 

It  would  seem  probable  that  proteins  may  yield  a  similar  poison  in 
whatever  way  their  hydrolysis  is  brought  about,  provided  the  cleav- 
age is  not  too  deep-seated.  For  example,  Rosenow^'  has  found  that 
pneumococci  and  other  bacteria,  permitted  to  autolyze  for  a  proper 
length  of  time,  produce  poisonous  substances  with  all  the  toxicologic 
characters  of  the  anaphylatoxin.  Too  extensive  autolj^sis  again  de- 
stroys the  poison,  which  is  also  produced  by  digestion  of  pneumo- 
cocci w'ith  serum  from  normal  guinea-pigs,  and  more  rapidly  with  serum 
from  sensitizeti  animals,  w'hich  likewise  causes  a  demonstrably  more 
rapid  proteolysis.  The  pneumococcus  anaphylatoxic  poison  is  soluble 
in  ether  and  seems  to  be  a  base,  containing  amino-acids,  but  Fried- 
berger chd  not  find  anaphylatoxin  made  from  serum  proteins  to  be 
soluble  in  ether  or  alcohol,  nor  was  it  precipitated  with  the  globulins. 
The  so-called  "Abderhalden  method"  of  sero-diagnosis  of  pregnancy, 
which  depends  on  the  presence  of  specific  proteolytic  properties  in  the 
blood,  is  an  especially  studied  instance  of  these  principles,  and  is 
discussed  later. 

Presumably  anaphylactic  intoxication  is  hut  an  exaggeration  of  the 
normal  process  of  defense  of  the  body  against  foreign  proteins  {including 

'^'^  Friedberger's  explanation  of  the  inhibiting  effect  of  salt  as  interference  with 
complement  action,  has  been  questioned.  (See  Zinsser,  Arch.  Int.  Med.,  1915 
(16).  238.) 

2-  See  Besredka  et  al,  Zeit.  Immunitat.,  1912  (16),  249. 

"  Jour.  Infec.  Dis.,  1912  (11),  94  and  235. 


196  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

bacteria)  through  digestion.  Normally  this  is  accomplished  in  the 
alimentary  tract,  and  complete  disintegration  past  the  toxic  stage  is 
made  certain  by  the  presence  of  erepsin  in  the  intestinal  wall;  but 
if  intact  foreign  protein  molecules  reach  the  blood  in  any  wa}-,  this  same 
digestive  destruction  is  performed  by  the  enzymes  of  the  blood  or 
tissues.  So  abnormal  is  the  "parenteral"  introduction  of  foreign 
proteins  that,  once  it  has  happened,  the  protective  mechanism  is 
stimulated  to  the  production  of  large  amounts  of  proteolj^tic  sub- 
stances, and  on  this  account  if  another  quantity  of  the  same  protein 
is  again  parenterally  introduced  the  breaking  down  of  the  protein  is 
extremely  rapid.  Certain  of  the  disintegration  products  are  toxic,  but 
with  the  normal  rate  of  disintegration  the  amount  present  at  any  one 
time  is  inadequate  to  cause  poisoning;  when  the  proteolysis  is  accele- 
rated, as  in  the  sensitized  animal,  a  poisonous  dose  may  be  produced, 
with  the  resulting  anaphylactic  intoxication. ^^  Whether  this  proteo- 
lysis takes  place  both  in  the  blood  and  tissues  is  not  known.  It  has 
been  found  that  the  specific  proteolytic  power  of  the  blood  is  increased 
in  sensitized  animals,  but  on  the  other  hand,  there  is  evidence  that 
without  the  intervention  of  the  livfer  (at  least  in  dogs)  anaphylactic 
intoxication  cannot  take  place  (Manwaring  and  others),^*  During 
the  reaction,  in  any  event,  products  of  protein  In'drolysis  appear  in 
the  blood  (Abderhalden),^'^  but  there  is  no  demonstrable  destruction 
or  binding  of  the  injected  foreign  protein. ^^ 

Among  possible  cleavage  products  of  proteins  which  may  be  the 
toxic  agent  in  anaphylaxis,  is  |3-imidazolylethylamine  ("histamine"), 
which  is  derivable  from  histidine,  and  which  produces  effects  resembling 
acute  anaphylactic  intoxication. 2''  Not  only  does  histamine  cause 
marked  fall  in  blood  pressure,  bronchial  spasm  in  guinea-pigs  and 
obstruction  to  the  pulmonary  circulation  in  rabbits,  but  also  when 
applied  locally  it  causes  marked  urticaria  resembling  closely  that  of 
anaphylaxis.  Methylguanidine  is  said  to  produce  somewhat  similar 
but  slighter  symptoms, 2**  and  to  protect  sensitized  animals  from 
toxic  doses  of  the  antigenic  protein. ^^  Other  amines  possibly  may 
be  involved.  (See  Chapter  iv,  Ptomains;  Chapter  xxi.  Pressor 
Bases.) 

The  relation  of  the  normal  toxicity  of  certain  foreign  sera  to  ana- 
phylactic intoxication  has  not  been  determined,  but  there  seem  to  be 

^''  Hcilner  (Zeit.  Biol.,  1912  (58),  333)  believes  that  tlie  anaphylactic  poisons 
are  substances  which  normally  are  destroyed  by  proteolysis,  but  that  in  the  sensi- 
tized animals  there  is  a  depressed  catabolism  which  prevents  tlieir  destruction. 

2^  Falls  found  that  a  larger  intoxicating  dose  must  be  injected  into  the  portal 
system  to  produce  the  same  cfTects  than  by  peripheral  injection.  (Jour.  Infect, 
Dis.,  1918  (22),  83.) 

"Zeit.  phy.siol.  C^hem.,  1912  (82),  109. 

"Sec  Barger,  '"i'he  Simpler  Nalural  liases,"  London,  1914,  ]h  30.' 

28  lleydc,  Cent.  f.  Bhysiol.,  1911  (2.5),  441;  1912  (20),  401. 

29  Burns,  Jour.  Bhysiol.,  1918  (52),  .\.\.\ix. 


ANAPnVLAMS  Oh'  ALIJ<:R(!Y  \'.)7 

both  (lofiiiilo  similarities  and  diffcrcncos/^"  which  havo  been  discussed 
by  Loewit;'"  chief  of  these  differences  is  the  alxsence  of  the  broiicliial 
spasm  with  puhnonary  empliysema  which  is  characteristic  of  anaiili}'- 
laxis  in  guinea-pigs. 

Tlie  nnajihyhictic  j^oison  wouhl  seem  to  be  after  the  order  of  the 
alkak)i(hd  poisons,  at  least  from  the  pharmacological  standpoint,  since 
it  protluces  its  effects  quickl}^,  and  these  effects,  no  matter  how  severe, 
are  strictl}'"  transitory,  passing  off  completely  in  a  few  hours,  which 
indicates  that  (hke  morphine,  strychnine,  etc.)  they  do  not  produce 
any  deep-seated  structural  alterations  in  the  tissues.  According 
to  Schultz^^  ^i^Q  chief  effects  are  directly  on  the  smooth  muscles. 
Such  anatomical  alterations  as  are  produced,  of  which  hemorrhages 
and  waxy  degeneration  of  the  voluntary  muscles  of  respiration'^  are 
most  noticeable,  are  ascribable  to  the  effect  on  respiration,  which  in 
the  guinea-pig  often  amounts  to  total  asphyxiation  through  spasm 
of  the  musculature  of  the  bronchioles  (Auer  and  Lewis)  ■'^^  with  pro- 
found permanent  emphysematous  distension  of  the  lungs.  This  effect 
is  peripheral,  and  is  inhibited  by  atropine. ''  Calcium  salts  also  reduce 
anaphylactic  reactions. '''  The  poisonous  fraction  obtained  from  pro- 
teins by  Vaughan's  method  resembles  anaphylatoxin  in  that  it  causes 
a  fall  in  blood  pressure  by  paralyzing  the  vasomotor  endings  in  the 
blood  vessels  (Edmunds'^).  It  also  produces  local  urticaria  when 
rubbed  into  the  skin  and  behaves  much  like  histamine,  with  which, 
however,  it  is  not  identical.  One  gram  of  casein  yields  enough  of 
Vaughan's  poison  to  kill  800  guinea-pigs,  and  the  poison  seems  to 
contain  most  of  the  aromatic  radicals  of  the  proteins.     There  is  also 

^^  It  has  been  found  that  organ  extracts  are  especially  toxic  to  animals,  but 
that  this  toxicity  may  be  suppressed  by  a  minute  dose,  for  a  few  minutes  later 
large  doses  can  be  injected  with  impunitj',  although  the  blood  of  the  animal  is 
highly  toxic  during  the  immune  period,  which  is  of  brief  duration.  This  condi- 
tion is  called  skepto-phylnxis.  (See  Lambert,  Ancel  and  Bouin,  Compt.  Rend. 
Acad.^  Sci.,  1911  (154),  21.)  Yaughan  reports  the  finding  in  normal  tissues  of 
substances  rcsemliling  his  "protein  poisons,"  which  perhaps  come  from  autolysis 
or  tissue  metabolism  and  mav  be  related  to  the  "primary  toxicity"  of  organ  ex- 
tracts. N.  R.  Smith  (Jour.  Lab.  Clin.  Med.,  1919  (4),  '517,  full  review)  would 
attribute  this  toxicity  to  the  inorganic  constituents  of  the  extracts,  especially 
the  phosphorus  compounds. 

"  Arch.  exp.  Path.  u.  Pharm.,  1913  (73),  1.  See  also  DeKruif  and  Eggerth, 
Jour.  Infect.  Dis.,  1919  (24),  505. 

32  Bull.  Hvg.  Lai).,  U.  S.  P.  H.  and  M.  H.  Service,  1912  (80),  1. 

"  See  Cent.  f.  Pathol.,  1912  (23),  945. 

^*  In  the  rabbit  the  effects  seem  to  be  produced  chiefl\'  by  spasm  of  the  pul- 
monary arteries  (Coca,  Jour.  Immunol.,  1919  (4),  219)  while  the  predominant 
hepatic  and  portal  effects  in  dogs  is  attributed  to  the  highlj'  developed  musculature 
of  their  hepatic  veins  bv  Simonds  (Jour.  Amer.  INIed.  Assoc,  1919  (73),  1437). 

^Mour.  Exp.  Med.,'  1910  (12),  151;  Schultz,  Jour.  Pharm.  and  Exp.  Ther., 
1913  (3),  299. 

3«Kastle,  Healy  and  Buckner,  Jour.  Infcc.  Dis.,  1913  (12),  127. 

"  Zeit.  Immunitiit.,  1913  (17),  105.  See  also  Underhill  and  Hendrix,  Jour. 
Biol.  Chem.,  1915  (22),  465. 


198  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

much  other  evidence  of  the  importance  of  the  aromatic  radicals  in 
anaphylaxis.^'"* 

Other  effects  of  the  anaphylactic  toxin  are  leucopenia,  local  and  gen- 
eral eosinophilia,^^  reduced  coagulability  of  the  blood, ^°  and  a  severe  fall 
of  temperature  unless  the  dose  of  antigen  is  very  small  when  the  tem- 
perature may  rise.'*^  The  antitrypsin  content  of  the  blood  is  not  in- 
creased in  the  anaphylactic  animal  (Ando'*').  Poisonous  substances 
similar  to  anaphylatoxin  appear  in  the  urine  during  the  anaphjdactic 
intoxication  (Pfeiffer).^^  As  with  other  poisons,  anaphylatoxin 
produces  different  symptoms  in  different  animals.^'*  In  dogs  the 
chief  effects  are  a  great  fall  in  blood  pressure, ■*•*  loss  of  coagulability 
of  the  blood,  hemorrhagic  enteritis,  but  no  bronchial  spasm.  In 
rabbits  the  heart  is  severely  affected,  while  in  guinea-pigs  there  is  a 
remarkable  lack  of  interference  with  the  heart,  so  that  it  beats  long 
after  respiration  ceases.  A  pressor  substance  has  been  found  in  the 
serum  of  intoxicated  guinea-pigs,  which  is  not  present  in  the  artificial 
anaphylatoxin  and  therefore  presumably  is  produced  in  the  body  of 
the  animal. ^^  In  man  the  symptoms  are  most  like  those  in  the  guinea- 
pig.  If  the  protein  is  injected  into  the  skin  of  a  sensitized  animal 
there  follows  a  severe  local  reaction — hyperemia,  edema,  even  necrosis, 
— indicating  that  in  this  specific  proteolysis,  poisons  are  formed  wliich 
have  a  profound  local  effect,  especially  on  the  blood  vessels.  Repeated 
anaphylactic  intoxication  may  result  in  structural  changes  in  the 
kidneys,  heart  muscle  and  liver  (Longcope*^).  Metabolism  studies 
may  show  an  increased  toxicogenic  destruction  of  protein,'*"  but  the 
increase  in  amino-acids  presumably  resulting  from  proteolysis  in  the 
sensitized  individual,  is  not  large  enough,  if  it  does  occur,  to  be  signi- 
ficant.^^ However,  in  anaphylaxis  in  guinea  pigs,  as  well  as  after 
peptone  poisoning,  there  is  a  considerable  increase  in  noncoagulable 
and  urea  nitrogen  in  the  blood,  as  well  as  a  slight  increase  in  amino 
nitrogen,  but  it  is  not  known  whether  this  comes  from  the  tissues 
or  from  the  antigen-antibody  reaction  in  the  blood. '*^" 

3»  See  Baehr  and  Pick,  Arch.  Exp.  Path.,  1913  (74),  73. 

'^Literature  by  Moschowitz,  New  York  Med.  Jour.,  Jan.  7,  1911;  Schlecht 
and  Schwenker,  Arch.  exp.  Path.  u.  Pharm.,  1912  (68),  163;  Deut.  Arch.  klin. 
Med.,  1912  (108),  405. 

'"See  Bulger,  .Jour.  Infect.  Dis.,  1918  (23),  522. 

*^  See  Vaughan,  et  al.,    Zeit.  Immunitat.,  1911  (9),  458. 

^^Zeit.  Inununitiit.,  1913  (18),  1. 

^'Zeit.  f.  Ininuinitat.,  1911  (10),  550. 

■"^  Probably  from  influence  upon  the  nerve  endings  of  the  vessels  (Pearce  and 
Eisenl)rey,  Jour.  Infec.  Dis.,  1910  (7),  565). 

"  llirschfeld,  Zeit.  Lninunitat.,  1912  (14),  4(i(). 

^«  Jour.  J':xp.  Med.,  1913  (18),  678;  1915  (22),  793;  al.so  Houghton,  Jour.  Im- 
munol., 191G  (1),  105;  1919  (4),  213.  Not  confirmed  by  Bell  and  Ilartzell,  Jour. 
Infect.  Dis.,  1919  (24),  618. 

■•'See  Major,  Deut.  Arch.  klin.  Med.,  1914  (116),  248. 

^8  See  Auer  anil  Van  Slvke,  Jour.  Kxp.  Med.,  1913  (18),  210;  Barger  and  Dale, 
Biochem.  .Jour.,  1914  (8),  '670. 

""See  Hisanobu;  Amer.  .lour.  Plivsiol.,  1920  (50),  357. 


ANAPHYLAXIS  OR  ALLERGY  199 

There  is,  however,  iimch  doubt  as  to  the  identity  of  the  process  of 
anaphylatoxin  formation  (as  it  occurs  when  antipon,  antibody  and 
complement  are  incubated  in  vitro)  and  the  process  of  anaphylactic 
intoxication.  In  the  first  place,  a  poisonous  character,  apparently 
identical  with  this  "anaphylatoxin"  may  be  given  to  serum  without 
the  use  of  any  specific  antibody  whatever;  merely  agitating  fresh 
serum  with  anv  finely  divided  foreign  material  that  offers  large  total 
surfaces,  such  as  kaolin,  agar,  or  starch,  is  suflficient,  as  also  is  treat- 
ment with  lipoid  solvents,  such  as  chloroform  (Jobling).  In  fact, 
merely  removing  the  fibrin  from  the  plasma  may  make  the  resultant 
serum  highly  toxic,  even  for  the  very  animal  from  which  it  came. 
Furthermore,  if  anaphylactic  shock  were  the  result  of  anaphylatoxin 
formation  in  the  sensitized  animal  through  the  reaction  of  antigen  with 
antibody  and  complement,  the  intoxication  should  occur  if  antibody 
and  antigen  are  injected  simultaneously  into  an  animal;  but  as  a  mat- 
ter of  fact  the  animal  receiving  antibody  in  passive  sensitization  will 
not  react  unless  the  antigen  is  injected  at  least  three  hours  after  the 
sensitizing  serum  is  injected."**  This  incubation  period  is  supposed 
to  be  required  for  the  anaphylactic  antibody  to  be  fixed  in  the  cells 
where  the  reaction  takes  place  (Otto),  and  perhaps, in  modification 
of  the  antibody  so  that  it  has  a  greater  affinity  for  the  antigen  than 
it  has  while  free  in  the  serum  (Weil)  ;^°  also  in  acquiring  the  capacity 
to  affect  the  cells  after  union  with  the  specific  antigen.  Finally,  the 
isolated  nonstriated  muscle  tissue  (uterus)  of  a  sensitized  animal  gives 
specific  reactions  when  brought  in  contact  with  the  specific  antigen,  no 
matter  how  thoroughly  the  animal's  blood  has  been  removed  from  the 
tissues;  whereas,  the  uterine  muscle  of  an  animal  injected  with  sensi- 
tizing immune  serum  only  one  hour  before  killing  does  not  react  when 
in  contact  with  specific  antigen.  Weil  disputes  the  toxic  nature  of  ana- 
phylaxis, even  in  the  intracellular  reaction,  which  he  calls  a  "cellular 
discharge."  He  holds  that  in  the  guinea-pig  the  cellular  reaction 
takes  place  chiefly  in  the  nonstriated  muscles,  while  in  dogs  the  reaction 
is  essentially  hepatic,  resulting  in  a  profound  congestion  of  the  liver 
and  consequent  fall  of  blood  pressure,  decreased  blood  coagulability 
from  hepatic  action,  and  the  increase  in  proteolytic  products  in  the 
blood  characteristic  of  all  acute  hepatic  injuries. ^^  Even  in  the 
guinea-pig,  however,  the  liver  is  affected  in  anaphjdactic  shock,  and 
Meinicke^-  adds  to  the  evidence  of  cellular  anaphylaxis  by  finding  that 
the  perfused  liver  of  sensitized  guinea-pigs  reacts  to  antigen  with  a 
marked  inhibition  of  its  capacity  to  form  urea  from  ammonium  lactate. 

Nevertheless,  the  formation  of  anaphjdatoxin  is  an  interesting  phe- 
nomenon which  may  well  be  of  importance  in  human  intoxications, 

*^  See  Weil,  Jour.  Med.  Res.,  1914  (30),  S7;  Jour.  Immunol.,  1916  (1),  109. 
^^  Jour.  Med.  Res.,  1915  (32),  107. 
"  Jour.  Immunol.,  1917  (2),  525. 
"  Zeit.  Immunitat.,  1918  (27),  489. 


200  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

even  if  it  is  not  the  essential  phenomenon  of  the  anaphylactic  intoxica- 
tion. So  readily  is  blood  serum  made  toxic  in  vitro  that  it  seems 
highly  probable  that  a  similar  development  of  toxicity  may  take  place 
in  the  body.  Jobling^^  has  found  that  intoxication  from  anaphyla- 
toxin  formation  seems  to  occur  when  kaolin  is  injected  intravenouslj' 
into  animals,  and  hence  it  is  quite- possible  that  the  presence  in  the 
blood  of  abnormal,  finely  divided  bodies,  such  as  precipitated  proteins, 
cellular  fragments,  even  bacteria,  may  mechanically  cause  anaphjda- 
toxin  formation  in  vivo  just  as  they  do  in  vitro.  It  is  necessary  to 
distinguish,  however,  between  the  symptoms  that  result  from  capil- 
lary embolism  and  true  anaphylaxis,  failure  to  do  this  undoubtedly 
having  caused  many  erroneous  conclusions.^^" 
(  Recentlj^  it  has  been  suggested  that  a  process  similar  to  anaphy- 
'  lactic  intoxication  is  responsible  for  traumatic  shock,  disintegration 
of  traumatized  tissue  proteins  being  the  source  of  the  toxic  agent. 
(Quenu  and  Delbet,  Cannon).^* 

The  mechanism  of  anaphylatoxin  formation  is  not  yet  under- 
stood but  there  is  no  lack  of  theories.  The  original  explanation  was  that 
anaphylatoxin  formation  by  specific  antisera  is  the  result  of  digestion 
of  antigen  in  vitro  by  the  action  of  complement  united  to  the  antigen 
by  the  immune  antibody.  For  the  formation  of  anaphylatoxin  by 
inert  finely  divided  particles  the  explanation  advanced  was  that  the 
highly  developed  surfaces  of  these  particles  either  activated  comple-. 
ment,  or  united  it  to  the  serum  proteins  so  that  it  digested  them. 
Jobling"  has  advanced  the  hypothesis  that  normal  serum  antifer- 
ments,  which  are  believed  by  him  to  be  lipoidal  in  nature,  are  bound 
by  the  particles  or  by  specific  precipitates,  so  that  the  complement  is 
free  to  attack  the  serum  proteins.  In  any  case,  it  is  now  generallj'' 
agreed  that  the  poisonous  substance  is  derived  chiefly,  if  not  ertirely 
from  the  serum  of  the  intoxicated  animal,  and  not  from  the  antigen,  even 
in  the  case  of  anaphylatoxin  formation  by  specific  antigen-antibody- 
complement  reactions. ^^  This  fact  would  seem  to  explain  why  the 
poison  seems  to  be  the  same,  as  far  as  we  can  analyze  it  by  phar- 
macological methods,  no  matter  what  protein  is  used  as  antigen,  or 
whether  produced  by  immune  or  by  nonspecific  reactions,  or  by 
chemical  means,  such  as  that  of  Vaughan. 

Jobling,  who  holds  to  the  importance  of  anaphylatoxin  formation  as  the  cause 
of  anaphylactic  intoxication,  presents  the  following  conception  of  anaphj'laxis: 
During  the  course  of  sensitization  there  occurs  the  mobilization  of  a  nonspecific 
protease,  which  is  greatly  increased  during  acute  anaphj'lactic  shock;  at  this  time 

"  Jobling,  Petersen  and  Eggstein,  Jour.  Exp.  Med.,  1915  (22),  590. 

''"See  Hanzlik  and  Karsner,  Jour.  Pharmacol.,  1920  (14),  379. 

"  C.  R.  Soc.  Biol.,  191S  (71),  850;  Rev.  d.  Chir.,  1919  (3S)  309. 

"  Zcit.  Immunitat.,  1911  (23),  71;  Jour.  Exp.  Med.,  1915  (22),  401. 

**  That  the  antigen  must  be  digestible,  however,  is  suggested  by  the  observa- 
tion of  Ten  Broeck  (Jour.  Biol.  Chem.,  1914  (17),  309)  that  proteins  racemized 
by  Dakin's  method,  which  cannot  be  digested  by  proteolytic  enzymes,  arc  unable 
to  cause  anaphylaxis. 


ANAPHYLAXIS  Oh'  ALLERGY  201 

there  is  also  a  decrease  in  antifennont  wliich  permits  proteolysis  of  the  animal's 
own  i)r()toins.  As  a  result,  there  is  to  l)e  founrl  an  increase  in  noncoaKiilable 
nitrofjicn  and  ainino-acids  of  the  hlood,  and  a  decrease  in  sorinn  proteases.  "The 
acute  intoxication  is  hrouglit  al)out  by  the  cleavage  of  serum  jjroteins  throiiRh  the 
pci)tone  sta}j;e  l)y  a  non-specific  protease.  The  specihc  elements  lie  in  the  rapid 
mobilization  of  this  ferment  and  tlie  colloidal  serum  changes  which  bring  about 
the  change  in  antifcrment  titer." 

As  a  result  of  extensive  studies,  Novy  and  DeKruif  have  developed  ideas  con- 
cerning the  nature  of  an:ii)hylaxis  quite  different  from  those  ordinarily  held.*^ 
lM)ll()wing  the  observations  of  Bordet,  and  otliers,  that  incubation  of  fresh  normal 
serum  with  agar  renders  it  capable  of  producing  symptoms  resembling  those  of 
anajihylaxis,  they  have  found  reason  to  believe  that  the  effects  of  bacteria  may  also 
depend  on  similar  phenomena,  rather  than  on  hypothetical  endotoxins.  As  little  as 
9  mg.  of  agar  may  produce  fatal  intoxication  if  injected  in  a  suitable  physical  state 
intravenously  into  a  guinea-pig.  As  perfectly  dissolved  'VA'itte  peptone  or  even 
distilled  water  also  produce  anaphylatoxin  when  mixed  with  serum,  it  seems  improb- 
able that  the  anaphylatoxin  formation  depends  on  surface  phenomena.  Apparently 
anaphylatoxin  formation  is  closely  a.ssociated  with  the  coagulation  of  the  blood, 
which  becomes  highly  toxic  in  the  early  stages  of  clot  formation.  Xo  evidence 
could  be  found  of  protein  cleavage  or  enz5mie  action  during  the  formation  of  ana- 
phylatoxin. It  is  thought  that  in  specific  anaphylaxis  the  substance  that  induces 
the  formation  of  anaphylatoxin  is  formed  by  the  interaction  of  the  antigen  with 
the  antibody.  The  process  of  anaphylatoxin  formation  parallels  closely  that  of 
fibrin  formation,  to  which  it  may  be  related.  Anaphylatoxin  is  believed  to  be  not 
a  proteose  but  a  larger  molecular  complex  than  serum  albumin,  possessing  certain 
globulin  characteristics.  It  is  associated  with  the  euglobulin  fraction  of  the 
,  serum.** 

These  and  many  other  observations  in  the  literature  support  the  idea  that  a 
change  in  the  degree  of  dispersion  of  the  blood  colloids  may  be  the  fundamental 
matter  in  anaphylaxis.*' 

The  Anaphylactic  Antibody  (Anaphylactin). — That  anaphylaxis, 
like  other  immunity  reactions,  depends  upon  the  presence  of  specific 
antibodies  in  the  blood  of  the  sensitized  animal,  is  shown  by  the  pro- 
duction of  passive  anaphylaxis  in  normal  animals,  by  injecting  into 
them  a  few  cubic  centimeters  of  blood  or  serum  from  a  sensitized  ani- 
mal. Such  animals  become  sensitive  in  a  few  hours  to  the  specific 
antigen,  no  matter  what  species  of  animal  furnishes  the  serum,  show- 
ing that  various  anaphylactins  can  unite  with  the  same  complement, 
although  strongly  specific  as  to  the  antigen.  In  active  sensitization 
the  anaphylactin  appears  in  the  blood  in  appreciable  quantities  about 
eight  days  after  the  sensitizing  injection,  increases  to  a  maximum 
between  the  15th  and  30th  days,  and  then  very  slowly  decreases.  The 
reaction  of  antibody  and  antigen  is  strictly  quantitative,  as  with  all 
amboceptor  reactions.  The  amount  of  antibody  developed  seems  to 
be  limited,  for  after  a  sensitized  animal  is  given  a  sub-lethal  intoxicating 
dose  of  protein  it  may  be  no  longer  sensitive  to  this  protein,  and  this 
refractory  or  anti-anaphylactic  condition  persists  for  three  weeks  or 
more.  It  has  been  demonstrated  especialh'  conclusively  by  Weil  and 
Coca,®"  that  this  refractory  condition  is,  as  Friedberger  suggested,  de- 

*Mour.  Infect.  Dis.,  1917  (20);  recapitulation  in  Jour.  Amer.  Med.  Assoc, 
1917  (68),  1524. 

"DeKruif  and  Eggerth,  Jour.  Infect.  Dis.,  1919  (24),  505.  They  state  also 
that  in  primarilv  toxic  sera  the  toxic  element  is  associated  wdth  the  pseudoglobulin. 

"  See  Kritciiewskv,  Jour.  Infect.  Dis.,  1918  (22),  101. 

60  Zeit.  Immunitat.,  1913  (17),  141. 


202  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

pendent  upon  saturation  or  exhaustion  of  all  the  anaphylactic  anti- 
bodies, and  hence  the  amount  of  these  antibodies  present  free  in  the 
blood  of  a  sensitized  animal  must  be  relatively  small,  for  a  few  milli- 
grams of  the  specific  protein  is  sufficient  to  saturate  them,  e.  g.,  the 
amount  of  antibody  present  in  3  c.c.  of  serum  from  a  guinea-pig  sen- 
sitized with  horse  serum  could  be  neutralized  with  from  0.0005  to 
0.01  c.c.  of  horse  serum. ^^  They  are,  however,  very  persistent,  re- 
maining in  guinea-pigs  through  the  entire  life  of  an  animal  sensitized 
when  young.  They  also  pass  from  the  mother  to  the  fetus,  conferring 
a  passive  sensitization  which,  like  passive  sensitization  from  injection 
of  serum  from  a  sensitized  animal,  is  of  relatively  brief  duration, 
in  contrast  to  the  persistence  of  active  sensitization.''-  Anaphylactin, 
like  amboceptor,  resists  heating  at  56°  for  one  hour,  and  is  salted  out 
from  serum  in  the  globulin  fraction. ^^  It  can  be  formed  in  animals 
made  leukopenic  with  thorium-X.^^  Friedberger  contends  that  it 
is  identical  with  the  precipitin,  a  view  yet  under  discussion,''^'*  but 
strongly  supported  by  Weil's  observations.^^ 

WeiP^  observed  certain  phenomena  which  led  him  to  conclude 
that  in  anaphylaxis  the  specific  antibody  must  be  largely  fixed  in  the 
cells,  and  that  it  is  in  the  cells  that  the  reaction  occurs;  apparently 
the  antibodies  present  in  the  blood  of  the  sensitized  animal  are  insuf- 
ficient to  protect  its  cells  from  the  foreign  protein,  hence  the  cellular 
intoxication.  In  support  of  this  idea  is  the  observation  of  Dale" 
that  the  isolated  smooth  muscle  of  sensitized  guinea-pigs  is  specifically 
sensitive  to  the  foreign  protein.  Weil  states  that  "all  the  evidence 
proves  conclusively  that  anaphylactic  shock  is  induced  by  reaction 
between  anchored  antibody  and  antigen,  and  that  circulating  anti- 
body plays  absolutely  no  role  in  its  production." 

The  anaphylactin  shows  quite  the  same  characteristics  of  specificity 
as  the  other  immune  antibodies,''^  in  that  proteins  of  closely  related 
species  tend  to  interact,  while  proteins  of  very  distinct  biological  or 
chemical  nature  are  easily  distinguished.  Thus,  guinea-pigs  sensi- 
tized with  ape  serum  will  react  with  human  scrum,  but  not  with  serum 
from  dog  or  ox  or  fowl.     However,  in  the  final  analysis,  the  speci- 

6'  Anderson  and  Frost,  Jour.  Med.  Res.,  1910  (23),  31. 

^2  The  brief  duration  of  passive  sensitization  presumably  depends  on  the  forma- 
tion of  antibodies  for  the  foreign  sensitizing  serum,  constituting  the  condition 
of  "antisensitization"  as  contrasted  with  the  refrac^tory  period  which  results  from 
the  exhaustion  of  antibodies  by  antigen.  (See  Weil,  Zeit.  Immunitat.,  1913  (20), 
199;  1914  (23),  1.)  • 

"^  However,  Schiff  and  Moore  state  that  in  immune  sera  the  albumin  fraction 
contains  both  the  agent  that  confers  passive  sensitization  ami  the  constituent 
that  causes  the  "primary  toxicity"  of  foreign  sera.  (Zeit.  immunitat.,  1914  (22), 
009.) 

"  Corper,  Jour.  Infect.  Dis.,  1919  (25),  248. 

"■"•See  Zins.ser,  .Jour.  l':xp.  Med.,  1912  (15),  529. 

"*  Jour.  Immunol.,  1910  (1),  1. 

6«  Jour.  Med.  Research,  1913  (27),  497;  1914  (30),  299-364;  1915  (32),  107. 

"Jour,  rharm.,  1913  (4),  107. 

08  See  review  in  Jour.  Infect.  Dis.,  1911  (S),  73. 


HAY  FEVER  203 

ficity  depends  upon  the  chemical  composition  of  the  antigenic  protein, 
rather  than  its  biological  origin,  for  I  have  found  it  possible  to  dis- 
tinguish in  the  hen's  egg  five  distinctly  different  antigens,  and  these 
correspond  to  five  proteins  which  have  been  distinguished  by  chemical 
measures.""  Together  with  Dr.  T.  B.  Osborne,  working  with  purified 
vegetable  proteins,  I  have  found  evidence  that  a  single  isolated  pro- 
tein (hordein  or  gliadin)  may  contain  more  than  one  antigenic  radical."" 
As  Osborne^^  has  said,  "chemically  identical  proteins  apparently  do 
not  occur  in  animals  and  plants  of  different  species,  unless  they  are 
biologically  very  closely  related."  Whether  the  chemical  differences 
that  determine  specificity  are  of  quantitative  nature,  which  can  be 
disclosed  by  analytic  means,  or  whether  they  are  sometimes  dependent 
upon  spatial  relationships  of  the  amino-acid  radicals,  as  Pick  sug- 
gests, remains  to  be  determined.  My  own  experience  indicates  that 
usually,  at  least,  proteins  distinguishable  by  anaphylactic  reactions 
also  show  readily  distinguishable  chemical  differences. 

HAY-FEVER 

In  1902  Dunbar"-  demonstrated  conclusively  that  typical  hay-fever,  in  its 
several  forms,  is  due  to  pollen  of  various  sources;  in  all,  twenty-five  varieties  of 
grass  and  seven  varieties  of  plants  of  other  sorts  being  found  whose  pollen,  when 
placed  upon  the  nasal  or  conjunctival  mucous  membranes  of  haj'-fever  patients, 
causes  a  typical  attack  of  the  disease.  In  Germany  the  disease  seems  to  come 
chiefly  from  pollen  of  the  grasses  and  grains  (rye  pollen  being  most  active),  whereas 
in  America  the  most  important  pollen  seems  to  come  from  members  of  the  Ambrosia 
(ragweed)"'  and  Granimaceae  (grasses).  Dunbar  also  found  that  the  toxic  con- 
stituent could  be  dissolved  from  the  pollen  in  salt  solution,  and  seemed  to  be  a 
protein. 

The  protein  constituents  of  the  pollen  of  rye  ha\e  been  studied  further  by 
Kammann,^^  who  found  three  proteins,  one  of  which,  an  albumin,  was  found  to 
contain  all  the  active  matter.  This  constitutes  about  5.5  per  cent,  of  the  entire 
weight  of  the  pollen,  is  weakened  but  little  by  heating  to  S0°,  and  is  not  destroyed 
by  boiling;  it  is  but  partly  destroyed  by  pepsin  and  trypsin,  and  resists  acids  but 
not  alkalies.  Analysis  of  pollen  from  Ambrosia  by  J.  H.  Koessler"^  showed  that 
most  of  the  nitrogen  present  was  protein  nitrogen,  with  14.72  per  cent,  arginine, 
14.05  per  cent,  histidinc  and  3.18  per  cent,  l.ysine.  Heyl'^  obtained  from  Ambrosia 
pollen  a  mixture  of  albumose,  peptone,  albumin  and  glutelin,  the  latter  being  most 
abundant.  A  solution  containing  O.OOS  mg.  of  pollen  protein,  which  amount  is 
contained  in  two  or  three  pollen  grains,  produf^es  a  reaction  in  susceptible  indi- 
\aduals,  but  large  amounts  have  no  effect  on  normal  persons. 

Dunbar  manufactured  an  "antitoxic"  serum  by  immunizing  horses  against 
the  pollen,  believing  that  he  was  dealing  with  a  toxin,  but  its  efficacy  is  more  than 

^'  The  chief  proteins  obtained  by  fractionating  serum  give  cross  reactions  with 
each  other,  but  strongest  \vith  the  homologous  protein  (Kato,  Mitt.  med.  Fak. 
Univ.  Tokio.  1917  (IS),  195. 

"0  Jour.  Infec.  Dis.,  1913  (12),  341. 

"' Harvey  Lectures,  1910-11. 

'-  Full  re^^ew  of  subject  and  literature  given  by  Prausnitz,  KoUe  and  Wasser- 
mann's  Handbuch,  1913  (2),  1469;  Koessler,  Forchheimer's  Therapeutics,  1914 
(5).  671. 

"'  See  Cooke  andVan  der  Veer,  Jour.  Immunol.,  1916  (1),  201;  Goodale,  Boston 
Med.  Surg.  Jour.,  1914  (171),  695. 

"^  Hofmeister's  Beitr.,  1904  (5),  346;  Biochem.  Zeit.,  1912  (46),  151. 

'5  Jour.  Biol.  Chem.,  1918  (35),  415. 

"«  Jour.  Amer.  Chem.  Soc,  1917  (39),  1470;  1919  (41),  670. 


204  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

doubtful.  The  processes  involved  in  hay  fever  are  characteristically  of  the  nature 
■  of  an  anaphylactic  reaction  to  a  foreign  protein,  in  which  case  we  cannot  speak  of 
either  a  toxin  or  an  antitoxic  senim.  The  hypersensitization  seems  sometimes 
to  be  established  spontaneously  throusrh  inheritance,  but  no  antibodies  can  be 
demonstrated  in  the  blood  of  sensitized  persons,  although  the  cells  of  the  skin 
and  mucous  membranes  are  reactive,"'  so  that  the  specific  protein  responsible  for 
the  trouble  may  be  determined  by  skin  or  conjunctival  tests.  Essentially,  there- 
fore, hay  fever  is  one  of  the  cases  of  hypersensitivity  to  foreign  protein,  of  the  same 
class  as  horse  asthma,  food  urticarias,  etc. 

THE  ABDERHALDEN  REACTION 

This  reaction  is  based  upon  the  hypothesis  that  the  animal  body  reacts  to 
the  presence  of  foreign  proteins  by  providing  specific  means  of  destroying  them 
through  proteolysis,  and  hence  is  fundamentally  the  same  as  the  anaphylaxis 
reaction  as  conceived  by  Vaughan,  Friedemann,  Friedberger  and  others.  It 
differs  from  the  other  reactions  of  this  class  merely  in  that  tlie  methods  used  for 
determining  the  proteolysis  are  chemical  rather  than  biological.  The  occurrence 
of  a  reaction  is  indicated  by  the  production  of  diffu.sible  products  of  protein  hy- 
drolysis, which  may  be  detected  by  any  one  of  several  methods,  although  most 
used  is  "ninhydrin"  (triketohydrindene  hydrate)'*  which  reacts  with  any  alpha- 
amino  acid,  the  resulting  condensation  compound  being  a  blue  or  violet  color;  or 
by  observing  the  change  in  optical  rotation  that  occurs  in  a  solution  of  peptone 
under  the  hydrolytic  action  of  the  serum. 

It  has  undergone  much  the  same  series  of  shifting  explanations  as  the  other 
reactions  of  this  class.  At  first,  like  the  other  proteolytic  reactions,  it  was  assumed 
that  the  antigen  was  digested;  but,  as  with  the  precipitin  and  anaphylaxis  reac- 
tions, evidence  was  found  by  numerous  observers  that  not  the  antigen  but  the 
proteins  of  the  immune  serum  are  the  chief  or  sole  source  of  the  cleavage  products. 
For  some  reason,  hard  to  explain,  it  has  always  been  referred  to  as  if  it  were  the 
result  of  the  formation  of  specific  enzymes  which  attacked  the  antigen,  in  spite 
of  the  repeated  demonstration  that  sera  giving  positive  reactions  can  be  inacti- 
vated by  heat  and  reactivated  by  normal  serum, '^  thus  throwing  it  into  the  class 
of  amboceptor-complement  reactions,  with  which  it  agrees  in  principle. 

Having  been  introduced  first  as  a  method  for  diagnosing  pregnancy,  on  the 
principle  that  in  pregnancy  the  chorionic  cells  of  the  placenta  enter  the  maternal 
circulation  and  as  foreign  proteins  cause  the  formation  of  specific  "defensive  fer- 
ments," it  was  at  once  taken  up  as  a  clinical  procedure,  and  as  a  result  an  enormous 
literature  on  this  reaction  was  rapidly  produced.  Much  of  this  represents  highly 
uncritical  work,  largely  from  workers  not  trained  or  experienced  in  immunological 
principles,  and  hence  it  is  not  profitable  to  review  it  in  extenso  here.  Abderhalden's 
own  views  are  given  in  full  in  his  monographs^"  and  there  exist  numerous  critical 
reviews.^'     The  status  of  the  reaction  at  this  writing  seems  to  be  as  follows: 

Animals,  or  man,  after  having  foreign  proteins  of  any  sort  enter  the  blood 
stream,  may,  and  commonly  do  show  an  altered  condition  of  their  serum,  whereby 
when  their  serum  is  incubated  with  the  antigen  under  suitalile  conditions  very 
minute  quantities  of  the  products  of  protein  cleavage  may  be  set  free,  and  recog- 
nized when  dialyzed  away  from  the  digesting  mixture;  or,  a  measuralile  change  in 
optical  rotation  of  the  digestion  mixture  occurs.  However,  jierfectly  normal 
sera  may  at  times  cause  a  similar  proteolysis,  usually  but  not  always  less  thaji  with 
the  immune  serum. 

The  digestion  seems  to  involve  chiefly  the  serum  jiroteins  rather  than  the  anti- 
gen, although  under  certain  conditions  there  may  he  some  digestion  of  the  antigen. 

"  See  Cooke,  Flood  and  Coca,  Jour.  Immunol..  1917  (2),  217 

'*  Concerning  the  mechanism  of  the  ninhydrin  reaction  see  Retinger,  Jour. 

Amer.  Chem.  Soc,  1917  (39),  1059. 

'9  See  Stephan,  Mlinoh.  med.  Woch., "1914,1(61),  801;  Ilauptman  ibid.,  p.  1107; 

Bettencourt  and  Monezes,  Compt.   Rend.  Soc.   l^iol.,   191()  (77),   102. 
*"  Emil  Al)der]ialdcn,  "Schutzfermcnte  des  tierischen  (_)rgaiusmus." 
81  See  Wallis,  Ouart.  Jour.   Med.,   1910  (9),   13S;  Bronfenbrenner,  Jour.  Lab. 

Clin.  Med.,  1915  (1),  79;  1910  (1),  573.     Hulton,  Jour.  Biol.  Chem.,  1910  (25), 

103. 


THE  AlWEIUIALDEN  REACTION  205 

Bronfenbrennor  holds  tfiat  the  enzymes  exhibit  no  selectivity,  digesting  both  the 
antigen  and  tlie  serum   impartially.** 

Apparently  the  digestion  is  accomplished  by  serum  complement,  or  at  least 
normal  serum  enzymes,  rather  than  by  any  new-formed  specific  enzyme,  although 
enzymes  set  free  from  the  tissues  have  been  held  responsible  by  some. 

The  mechanism  of  the  reaction  is  not  understood.  Jol>ling  and  Petersen 
have  suggested  that  the  antigen-antibody  combination  may  adsorb  or  bind  the 
antiproteases  of  the  serum,  so  that  the  normal  protease  digests  the  serum  proteins. 
Or  it  niay  be  that  union  of  antigen  and  antibody  activates  the  complement,  or 
binds  it  to  the  antibodj'  so  that  it  digests  either  the  antibody  or  other  proteins 
of  the  serum.  It  also  is  suggested  that  enzymes  are  set  free  from  the  tissues 
injured  by  the  specific  protein,  or  bj'  disease,  which  digest  the  foreign  protein  or  the 
cellular  proteins  that  may  have  escaped  from  the  tissues  into  the  l)lood  stream. 

The  reaction  possesses  a  certain  specificity,  but  just  the  degree  of  this  specificity 
has  not  been  agreed  upon.  The  claim  of  Abderhalden*^  and  his  followers,  that  it  is 
by  far  the  most  specific  of  immunity  reactions,  whereby  disintegration  of  small 
amounts  of  any  given  organ  of  an  individual  can  be  determined  by  specific  reactions 
between  his  serum  and  that  organ,  with  such  refinement  that  even  cerebral  localiza- 
tion is  possible,*^  is  diflficult  to  accept.  There  are  so  many  possible  sources  of  error  in 
the  original  technic  that  even  with  great  care  the  charge  of  incorrect  results  from 
incorrect  technic  cannot  be  escaped,  and  therefore,  those  who  do  not  accept  the 
doctrine  of  its  specificity  are  always  on  the  defensive.  Nevertheless,  so  many  care- 
ful and  experienced  investigators  have  found  the  original  Abderhalden  reaction  to 
give  at  times  absoluteh^  non-specific  and  hopelessly  paradoxical  results,  that  its 
diagnostic  value  for  either  clinical  or  scientific  purposes  must  be  considered  at 
present  as  unproved,*^  whatever  the  final  decision  as  to  its  standing  as  a  specific 
reaction  may  be. 

Serum  treated  with  various  inert,  finelj'  divided  particles,  such  as  kaolin,  starch, 
silicates,  etc.,  may  acquire  the  property  of  giving  positive  reactions.  This  is 
another  point  of  resemblance  to  anaphylatoxin  formation,  and  against  the  speci- 
ficity of  the  reaction,  indicating  that  the  antigen  merelj^  acts  as  a  non-specific 
adsorbent. 

By  far  the  most  satisfactory  results  have  been  recorded  in  the  diagnosis  of 
pregnancy  by  means  of  placental  antigen.  This  may  be  explained  by  the  fact 
that  the  protease  activity  of  the  serum  seems  to  be  increased  in  pregnancy,*^  and 
hence  the  reaction  with  placenta  is  more  marked  than  with  the  serum  of  non- 
pregnant individuals.  But  simply  shaking  normal  serum  with  kaolin  or  other 
foreign  substances  may  cause  it  to  give  strong  reactions  with  placenta  antigen 
(Wallis). 

*- Supported  by  Smith  and  Cook,  Jour.  Infect.  Dis.,  1916  (18),  14.  De  Waele 
states  that  it  is  the  serum  globulin  that  is  digested  (Compt.  Rend.  Soc.  Biol.,  1914 
(76),  627). 

*'  A  reply  to  numerous  criticisms  is  given  by  Abderhalden,  Ferment forschung, 
1916  (1),  351;  this  and  other  numbers  of  this  journal  also  consist  largely  of  articles 
on  the  Abderhalden  reaction. 

8^  See  Retinger,  Arch.  Int.  Med.,  1918  (22),  234. 

*^  O.  J.  Elsesser  (Jour.  Infect.  Dis.,  1916  (19),  655),  working  in  my  labora- 
tory with  the  purified  vegetable  proteins  of  Osborne,  found  that  at  best  the  speci- 
ficitj'  of  the  reaction  was  less  than  that  of  the  anaphylaxis  reaction,  and  there 
were  many  absolutely  non-specific  and  irrational  reactions.  As  these  pure  proteins 
furnish  a  much  more  appropriate  material  for  studying  specificity  than  the  tissues 
or  sera  commonly  used,  it  would  seem  that  the  results  thus  obtained  are  excellent 
proof  of  the  uncertainty  and  unreliability  of  the  reaction.  Careful  quantitative 
studies  of  the  setting  free  of  amino-acids  by  serum  incubated  with  placenta, 
by  Van  Slyke  and  his  associates,  also  showed  a  complete  lack  of  specific  proteolysis 
by  pregnancv  serum  (Arch.  Int.  Med.,  1917  (19),  56;  Jour.  Biol.  Chem.,  1915 
(23),  377;  see" also  Hulton,  ibid.,  1916  (25),  163). 

«6  See  Sloan,  .Amer.  Jour.  Physiol.,  1915  (39),  9. 


CHAPTER  IX 

CHEMISTRY   OF   THE   IMMUNITY   REACTIONS    (Continued)— 

BACTERIOLYSIS,   HEMOLYSIS,   COMPLEMENT  FIXATION, 

AND   SERUM   CYTOTOXINS 

SERUM  BACTERIOLYSISi 

The  bactericidal  property  of  serum  may  be  shown  by  its  destruc- 
tion of  the  life  manifestations  of  bacteria  without  marked  alteration 
in  their  structure,  or  it  may  be  accompanied  by  dissolution  of  the 
bacterial  cell  (bacteriolysis).  How  much  of  the  bacteriotytic  process 
is  performed  by  the  serum  itself,  or  how  much  by  the  autolytic  en- 
zymes of  the  bacterial  cell,  is  unknown,  but  the  latter  is  probably 
a  factor.  The  bactericidal  property  of  immune  serum  has  been  shown 
to  be  quite  independent  of  the  antitoxic  properties  and  also  to  have 
quite  a  different  mechanism.  This  last  is  shown  in  the  following 
manner : 

If  we  heat  bactericidal  serum  made  by  immunizing  an  animal  against 
bacteria,  say  the  cholera  vibrio,  at  55°  for  fifteen  minutes,  it  will  be 
found  to  have  lost  its  power  of  destroying  these  organisms. ^  Normal 
serum  of  non-immunized  animals  is  equally  without  effect  upon  the 
vibrios.  If  however,  we  add  to  the  inactivated  heated  serum  an 
equal  quantity  of  inactive  normal  serum,  the  mixture  will  be  found 
to  be  as  actively  bactericidal  as  the  original  unheated  immune 
serum.  This  phenomenon  is  interpreted  to  mean  that,  by  immuniza- 
tion, some  new  substance  has  been  developed  which,  although  b}-  itself 
incapable  of  destroying  bacteria,  is  able,  when  united  with  some  sub- 
stance present  in  normal  serum,  to  destroy  bacteria  readily.  The 
substance  present  in  normal  serum  is  also  incapable  of  affecting  bac- 
teria by  itself,  but  needs  the  presence  of  the  substance  developed  by 
immunizing  to  render  it  bactericidal.  Hence  the  bactericidal  prop- 
erty in  this  case  depends  on  two  siibstances  acting  together:  one,  de- 
veloped during  immunization  and  therefore  called  the  immune  body, 
is  specific  for  the  variet}^  of  bacteria  used  in  immunization,  and  is  not 
destroyed  by  heating  at  55°.  The  other,  present  in  normal  serum,  is 
not  increased  during  immunization,  is  not  (altogether)  specific  in 
character,  and  is  destroyed  by  heating  at  55°;  as  its  action  is  com- 

^  Review  and  bibliography  by  Miiller,  Oppenheimer's  Handb.  d.  Biochcni .,  1909 
11(0,629. 

^  Normal  human  serum  often  exhil)its  some  jiower  to  destroy  Ijacteria,  even  after 
heating  to  55°.  The  nature  of  this  thermostable  bactericidal  agent  is  unknown. 
(See  Salter,  Zeit.  Hyg.,  1918  (86),  313). 

206 


AMBOCEPTOR  AND  COMPLEMENT  207 

plenientary  to  that  of  the  specific  iininune  body,  it  is  called  tlic  com- 
plement.^ 

It  is  believed  that  the  action  of  these  substances  is  as  follows:  The 
ininiunc  body  is,  like  antitoxin,  a  cell  receptor  which  unites  the  bac- 
teria to  the  cell.  It  differs  from  the  antitoxin,  however,  in  that  it  has 
two  affinities,  one  for  the  complement  and  the  other  for  the  bacterial 
substance.  On  account  of  the  existence  of  the  two  affinities  it  is  called 
an  amhoceptor.  Some  serums  contain  such  amboceptors  for  certain 
bacteria  without  previous  immunization,  hence  the  term  im^mune 
amboceptor  is  reserved  for  amboceptors  developed  by  imnmnization. 

Amboceptor  and  Complement.'* — The  function  of  the  ambo- 
ceptor is  to  unite  the  bacterial  protoplasm,  to  which  it  is  attached  by 
one  affinity,  to  the  complement  which  it  holds  by  its  other  affinity,  or, 
to  put  it  in  a  more  strictl}^  chemical  way,  the  addition  of  the  ambocep- 
tors to  the  bacteria  gives  them  a  chemical  affinity  for  complement. 
It  is,  therefore,  an  intermediary  body,  uniting  the  complement  to  the 
bacterial  protoplasm.  The  complement^  is  the  substance  that  actually 
destroys  the  bacteria,  in  which  respect,  as  well  as  in  its  susceptibility 
to  heat,  it  resembles  the  enzymes.  Complement  is  present  in  normal 
serums,  and,  as  it  is  not  increased  in  amount  during  immunization 
it  may  not  be  sufficient  to  satisfy  all  the  amboceptors,  hence  it  may 
be  impossible  to  secure  marked  bactericidal  effects  even  when  many 
amboceptors  have  been  formed.  If  the  complement  in  an  immune 
serum  has  been  destroyed  by  heating,  it  may  be  replaced  by  adding 
normal  serum  from  another  animal,  even  of  some  other  species;  indi- 
cating either  that  the  complement  is  not  absolutely  specific  in  its 
nature,  or  that  quite  the  same  complement  may  be  present  in  the 
blood  of  many  different  animals.  The  origin  of  the  complement  is 
unknown,  but  it  has  been  urged  that  the  leucocj^tes  are  an  important 
source  of /this  substance,  if  not  its  chief  one;^  there  is  evidence,  how- 
ever, that  various  organs  and  cells  may  also  produce  complement.'^ 
Its  most  important  characteristics  are  its  extreme  susceptibility  to 
heat,  and  the  resemblance  of  its  action  to  the  action  of  enzymes.^ 
Hektoen^  found  that  it  could  be  made  to  unite  with  Mg,  Ca,  Ba,  Sr, 
and  SO4  ions,  which  rendered  the  complement  (for  typhoid  bacilli  and 
red  corpuscles)  inactive.     Man  waring  ^°  found  that  these  ions  could  be 

^  The  polynuclear  leucocytes  also  contain  bacteriolj^tic  agents,  "  endolysins, "  of 
a  similar  complex  structure,  but  quite  distinct  from  the  serum  bacteriolvsins  (See 
Kling,  Zeit.  Immunitat.,  1910  (7),  1). 

■•  See  also  Hemolysis,  Chapter  X. 

°  Review  and  bibliography  by  Noguchi,  Biochem.  Zeit.,  1907  (6),  327. 

^  Cholera  antiserum  will  produce  the  Pfeiffer  phenomenon  of  lysis  of  cholera 
vibrios  in  animals  made  leucocj'te-free  with  thorium.  (Lippmann,  Zeit.  Immuni- 
tat., 1915  (24),  107.) 

'  See  Dick,  Jour.  Infect.  Dis.,  1913  (12),  HI;  and  Lippmann  and  Plesch,  Zeit. 
Immunitat.,  1913  (17),  548. 

«  See  Walker,  Jour,  of  Physiol.,  1906  (33),  p.  xxi. 

3  Trans.  Chicago  Path.  Soc,  1903  (5),  303. 

10  Jour.  Infectious  Diseases,  1904  (1),  112. 


208  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

separated  again  from  the  complement  by  simple  chemical  precipita- 
tion. Acids  stronger  than  CO2  and  of  the  higher  saturated  or  un- 
saturated fatty  acid  series,  inactivate  complement  in  strengths  greater 
than  n/40,  and  alkalies  are  equally  inhibitive.^'  Ultraviolet  rays 
destroy  complement. ^^  Sherwood^^  has  made  a  study  of  various  sub- 
stances that  may  be  present  in  the  blood  in  excessive  amounts  during 
pathological  conditions,  such  as  CO2,  lactic  acid,  acetone,  etc.,  and 
finds  that  they  interfere  seriously  with  the  action  of  complement, 
which  suggests  that  they  may  favor  infection  or  interfere  with  recovery 
from  infection. 

Presumably  the  complement  is  a  protein,  for  it  has  antigenic  prop- 
erties, so  that  immunization  with  sera  containing  either  complement 
or  complementoid  causes  anticomplement  activity  in  the  blood  of  the 
immune  animal.  Also,  it  is  destroyed  by  trypsin  free  from  lipase,  ^^ 
and,  like  other  colloids,  is  readily  adsorbed  by  surfaces;  hke  enzymes, 
complement  is  destroyed  by  shaking,  ^^  and  gradually  disappears  on 
standing.  There  are  some  striking  resemblances  between  the  be- 
havior of  complement  and  of  certain  compounds  of  protein  with  soaps 
and  lipoids,  as  pointed  out  especially  by  Noguchi,  but  that  these  are 
identical  with  true  complement  is  doubtful.  (See  Hemolysis.)  Its 
colloid  nature  is  attested  by  the  large  loss  when  complement  is  filtered 
through  Berkefeld  filters. ^*^ 

A  careful  review  of  the  evidence  has  led  Liefmann^'^  to  the  conclu- 
sion that  the  reaction  of  complement  to  sensitized  corpuscles  is  more 
hke  that  of  ferment  to  substrate  than  of  antigen  to  antibod3^  In  its 
effect  of  dissolving  bacteria  (and  also  other  cells  against  which  animals 
may  have  been  immunized)  co77iplement  resembles  the  enzymes,  and 
by  many  it  is  looked  upon  as  related  to  them,  but  the  changes  it  pro- 
duces do  not  resemble  those  produced  by  proteolj'tic  enzymes  in  all 
details.^*  In  particular,  complement  seems  to  participate  in  reactions 
according  to  the  law  of  definite  proportions,  unlike  the  enzymes.  ^^  In 
certain  immune  reactions,  colloids  (lecithin,  silicic  acid)-"  can  play  the 
role  of  complement  and  immune  body,  but  these  reactions  are  pro- 
bably quite  different  from  those  of  bacteriolysis  by  immune  serum. 

Structure  of  Complement. — According  to  the  Ehrlich  theory,  complement, 
like  toxins  and  enzymes,  possesses  at  least  two  groups:  one,  the  haptophore,  by 

11  Noguchi,  Biochem.  Zeit.,  1907  (6),  172. 

12  Courmont  et  al,  C.  R.  Soc.  Biol.,  1913  (74),  1152. 
"  Jour.  Infect.  Dis.,  1917  (20),  185. 

1^  Michaelis  and  Skwirsky,  Zeit.  Immunitat,.  1910  (7),  497. 

lii  Noguchi  and  Bronfenbrenner,  Jour.  Exp.  Med.,  1910  (13),  229;  Ritz,  Zeit. 
Immunitiit.,  1912  (15),  145. 

i»  See  Schmidt,  Arch.  f.  Hyg.,  1912  (70),  284;  Jour.  Ilyg.,  1914  (14),  437. 

1^  Zoit.  Immunitiit.,  1913  (16),  503. 

"  The  curve  of  complement  action  resembles  that  of  enzyme  action.  (Thicle 
and  Emblcton,  Jour.  Path,  and  Bact.,  1915  (19),  372.) 

i»  See  Liebermann,  Dcut.  med.  Woch.,  190(5  (32),  249. 

20  Landsteinor  and  Jagic,  Wien.  klin.  Woch.,  1904  (17),  03;  Munch,  med. 
Woch.,  1901  (51),  1185. 


CYTOTOXINS  209 

which  it  unites  witli  tho  .aiiihocrnptor;  the  other,  the  toxophoro  (or  zymophorr, 
because  of  its  enzynie-lilce  action),  which  attacks  the  bacterial  protoplasm.  It 
may  def^enerate  and  lose  its  toxoi)hore  f^roup  while  retaining  the  pow(!r  to  combine 
by  means  of  its  ha])topiiore  )j;i"'Jiil>»  thus  formiiip;  a  romplruicntoid.  (Complement 
and  aiubocejitor  exist  side  l)y  side  in  the  serum,  not  uniting  with  one  another 
until  the  ambocei)tor  has  become  attached  to  the  liactcrial  protoplasm. 

It  is  generally  stated  that  if  serum  containing  complement  be  so  treated  as  to 
separate  the  globulins  from  the  albumin,  it  is  found  that  the  complement  has  been 
divided  into  two  parts,  one  i)resent  in  each  of  the  protein  fractions.  The  globulin 
fraction  of  the  complement  will  unite  to  am])oceptor  which  is  fixed  to  cells,  and 
hence  is  called  the  mid-pircc  of  the  complement,  for  it  will  unite  also  with  the 
end-piece  of  the  complement  contained  in  the  all)umin  fraction,  and  then  cytolysis 
can  take  jjlace.  Without  the  intervention  of  the  globulin  mid-piece  the  albumin 
end-piece  cannot  unite  with  the  amboceptor,  while  in  the  ab.sence  of  end-piece 
the  amboceptor  mid-piece  complex  can  cause  no  cytolysis.  Both  fractions  of  the 
complement  are  destroyed  by  heat,  but  if  the  mid-piece  is  bound  to  the  ambo- 
ceptor it  resists  heating.  The  mid-piece  corresponds  to  Ehrlich's  haptophore, 
the  end-piece  to  the  toxophore  group,  and  this  complex  structure  is  common  to 
both  bacteriolytic  and  hemolytic  complement.  Bronfenbrenner  and  Xoguchi,^* 
however,  contend  that  the  supposed  cleavage  of  complement  is  merely  an  inactiva- 
tion  by  the  agencies  employed,  all  the  complement  being  in  the  albumin  fraction 
in  a  condition  capable  of  reactivation,  not  only  by  globulin  but  by  simple  ampho- 
teric substances,  a  view  which  has  not  been  generally  accepted. ^- 

Amboceptors  are  formed,  according  to  Wassermann,  and  Pfeiffer  and  Marx,  in 
the  spleen  and  hemopoietic  organs,  since  in  immunization  they  can  be  demonstrated 
in  these  organs  before  the)^  appear  in  the  circulating  blood.  The  stability  of  the 
amboceptors  is  very  considerable:  serum  prepared  in  1895  by  PfeifTer  against 
cholera  vibrios  was  found  to  have  lost  almost  none  of  its  activity  after  eight  years 
in  an  ice-box  (Friedberger).  Heating  twenty  hours  at  60°  scarcely  injures  them, 
but  70°  for  one  hour  destroys  them  almost  completely,  and  heating  the  serum  to 
100°  destroys  all  the  immune  bodies.  They  are  quite  resistant  to  putrefaction, 
and,  like  the  antitoxins,  do  not  dialj'ze.  Strong  salt  solutions  will  prevent  the 
union  of  complement  and  amboceptor  in  vitro,  and  probably  to  greater  or  less 
degree  in  the  animal  body,  but  the  union  of  antigen  and  amboceptor  is  not  pre- 
vented by  salt.^'  Alkalies  may  prevent  the  union  of  amboceptor  with  the  cells, 
or  extract  it  from  the  cell  to  which  it  has  united;  and  they  maj"-  also  inhibit  the 
union  of  amboceptor  and  complement.  Amboceptors  are  not  inactivated  by 
shaking,  as  is  complement,  but  they  are  destroyed  alike  by  ultraviolet  rays,  and 
both  resist  x-rays.^'* 

According  to  Pfeiffer  and  Proskauer,^^  digestion  of  the  globulin  precipitate,  in 
which  amboceptors  are  carried  down,  does  not  destroy  their  activity  completely 
even  when  all  the  proteins  are  thus  removed.  Removal  of  the  nucleo-albumin  or 
nuclein  does  not  remove  the  amboceptors  from  the  serum.  Immune  serum  kept 
three  months  in  alcohol  yielded  an  extract  with  distilled  water  that  was  rich  in 
immune  bodies,  but  almost  free  from  protein.  Pick,  Rhodain,  and  Fuhrmann 
found  that  immune  bodies  are  precipitated  entirely  in  the  euglobulin  fraction  of 
the  serum  protein.  From  these  experiments  it  has  been  thought  bj^  some  that 
the  bacteriolytic  amboceptor  is  not  itself  a  protein,  although  closely  associated 
with  the  serum  globulins.-^ 

CYTOTOXINS 

Just  as  precipitins  can  be  obtained  for  proteins  derived  from  other 
sources  than   bacterial   cells,   so  also  upon   immunizing  an   animal 

-^  Jour.  Exp.  Med.,  1912  (5),  598;  good  review  of  literature. 

"  See  Leschlev,  Zeit.  Immunitat.,  1916  (25),  44. 

"  Angerer,  Zeit.  Immunitiit.,  1909  (4),  243. 

"  Scaffidi,  Biochem.  Zeit.,  1915  (69),  162. 

"  Cent.  f.  Bakt.,  1896  (19),  191. 

^®  -\scoli  found  that  the  active  substance  of  anthracidal  serum,  which  is  not  an 
amboceptor,  is  contained  in  the  pseudo-globulin  fraction  of  asses'  serum,  but  in 
goat's  serum  part  is  in  the  euglobulin  fraction.     (Biochem.  Centr.,  1906  (5),  458.) 

14 


210  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

against  various  types  of  cells  other  than  bacteria,  substances  appear 
in  its  serum  that  exercise  a  destructive  effect  upon  the  tj^pe  of  cells 
injected.  In  other  words,  the  reactions  of  animals  to  infection  are 
not  specially  devised  for  combating  bacteria  and  their  products,  but 
can  be  equally  exerted  against  non-bacterial  cells  and  their  products. 
In  the  case  of  soluble  proteins,  as  before  mentioned,  the  antibodies 
show  their  effects  by  precipitating  them,  with  agglutination  of  the  par- 
ticles into  flocculi  and  perhaps  a  subsequent  digestion;  in  the  case  of 
cells,  whether  bacterial  or  tissue  cells,  the  antibodies  cause  agglutina- 
tion and  loss  or  impairment  of  vitality.  This  injury  may  be  mani- 
fested by  loss  of  motion  in  the  motile  cells  (bacteria,  spermatozoa, 
ciliated  epithelium)  or  by  solution  of  their  contents  (bacteriolysis, 
erythrocytolysis,  leucocytolysis,  etc.),  or  by  cell  death  without  marked 
morphological  alterations  (B.  typhosus,  spermatozoa).  If  we  inject 
red  corpuscles,  leucocytes,  spermatozoa,  renal  epithelium,  or  anj^  other 
foreign  cell,  the  reaction  is  as  specific  as  it  is  if  we  inject  bacteria,  and 
of  exactly  the  same  nature.  Therefore,  all  that  has  been  said  pre- 
viously concerning  bactericidal  substances  and  agglutinins  can  be 
transposed  to  apply  to  immunity  against  tissue  cells.  As  a  matter  of 
fact,  however,  the  transposition  is  generally  made  in  the  other  direc- 
tion, for  red  corpuscles  are  much  easier  cells  to  study  than  bacteria, 
because  their  laking  gives  prompt  and  readily  recognized  evidence 
that  the  toxic  serum  has  brought  about  changes.  Much  of  our  knowl- 
edge of  bactericidal  serum  has  been  obtained  through  studies  of  the 
mechanism  of  erythrocytolysis,  the  results  of  which  have  then  been 
applied  to  the  subject  of  bacteriolysis.  Both  on  this  account,  there- 
fore, and  because  solution  of  red  corpuscles  is  of  itself  an  important 
process  in  many  intoxications  and  diseases,  the  subject  is  of  great 
theoretical  and  practical  importance. 

Hemolysis-'  or  Erythrocytolysis 

In  hemolysis  the  essential  phenomenon  consists  in  the  escape  of 
the  hemoglobin  from  the  stroma  of  the  corpuscles  into  the  surroimd- 
ing  fluid.  As  it  is  not  exactly  known  in  what  way  the  stroma  holds 
the  hemoglobin  normally,  whether  purely  physically  or  in  part  chem- 
ically, or  whether  the  stroma  consists  of  a  spongioplasm  or  of  sac-like 
membranes,  or  both,  the  ultimate  processes  that  permit  the  escape  of 
the  hemoglobin  are  not  finally  solved.  However,  the  agents  bj'  which 
the  escape  is  brought  about  are  well  known  and  extensively  studied, 
and  they  are  found  to  be  of  extremely  various  natures.  Thej'  may 
be  roughh'  classified  as:  (1)  known  physical  and  chemical  agents;  (2) 
unknown  constituents  of  blood-serum;  (3)  bacterial  products;  (4) 
certain  vegetable  poisons;  (5)  snake  venoms. 

^'  Through  usage  this  term  has  been  limited  to  the  sohition  of  the  rod  corpus- 
cles, which  is  more  accuratelv  described  hv  the  term  crythron/toli/xis.  For  liihli- 
ography  see  Sachs,  Ergel)nisse  der  Pathol.",  1<»02  (7),  7i4;  Ht'Oti  (11),  515;  Kolle 
and  Wassermann's  Handbuch,  1913  (II),  793;  Landsteiner,  Handbuchd.  Biochem., 
1909  (II  (D),  395. 


MKCIIAMSM  OF  IIKMOI.YSIS  211 

Hemolysis  by  Known  Chemic\l  and  Physical  Agencies 

The  Mechanism  of  Hemolysis — -If  distilled  water  is  added  to 
corpuscles  of  any  kind,  osmotic  changes  arc  l)ound  to  occur,  since 
within  tlic  cells  arc  abundant  salts,  soluble  in  water,  whicii  will  begin 
to  diffuse  outward  in  an  attempt  to  establish  osmotic  equilibrium  be- 
tween the  corpuscles  and  the  surrounding  fluid.  Conversely,  water 
enters  the  corpuscles  at  the  same  time,  and  accumulating  there  leads 
to  swelling  until  such  injury  has  been  produced  as  permits  the  hemo- 
globin to  escape  and  enter  the  surrounding  fluid.  Before  this  oc- 
curs the  fluid  is  opaque  because  of  the  obstruction  to  light  offered 
by  the  red  cells,  but  on  the  completion  of  hemolysis  the  fluid  becomes 
transparent.  The  stroma  now  settles  to  the  bottom,  while  the  hemo- 
globin diffuses  into  the  fluid,  making  it  red,  but  perfectly  transparent. 
This  process  has  long  been  known  as  the  "laking"  of  blood,  and  is 
essentially  the  condition  present  in  all  forms  of  hemolysis.  That  the 
hemoglobin  escapes  only  through  inj\ny  of  the  stroma  and  not  through 
simple  osmotic  diffusion,  is  shown  by  the  fact  that  if  salt  solution  of 
the  same  concentration  as  normal  serum  is  used  instead  of  distilled 
water,  no  such  escape  of  hemoglobin  occurs.  As  hemoglobin  is  per- 
fectly soluble  in  salt  solution,  it  should  pass  out  if  it  diffused  as  do  the 
salts.  Since  there  is  no  escape  of  hemoglobin  in  such  a  salt  solution, 
it  is  evident  either  that  the  stroma  is  not  permeable  to  hemoglobin,  or 
else  the  hemoglobin  is  in  some  way  attached  to  or  combined  with 
the  stroma.  Again,  if  the  corpuscles  are  placed  in  a  solution  of  salt 
more  concentrated  than  their  own  fluids,  water  escapes  and  the  cor- 
puscles shrink;  as  no  hemoglobin  escapes  with  the  water,  it  is  evident 
that  the  stroma  is  not  permeable  to  hemoglobin  when  intact.  Because 
of  the  resemblance  of  the  process  of  hemolysis  to  the  rupture  of  plant 
cells  with  escape  of  their  contents  when  they  are  placed  in  distilled 
water,  it  might  be  assumed  that  hemolysis  is  largely  a  phj'sical  matter, 
but  if  a  red  corpuscle  in  an  isotonic  solution  is  cut  into  pieces,  the 
hemoglobin  does  not  escape,  indicating  that  its  structure  is  quite 
dissimilar  to  that  of  the  simple  vegetable  cell  and  that  there  is  some 
union  of  stroma  and  of  hemoglobin,  either  physical  or  chemical.-^ 
Physico-chemical  studies  also  indicate  that  there  is  no  true  covering 
membrane  to  red  corpuscles,  for  the  absorption  of  ions  by  hemoglobin 
is  the  same  as  the  absorption  by  corpuscles.-^  M.  H.  Fischer'"^ 
interprets  hemolysis  as  a  separation  of  lipoid-protein  stroma  and  ad- 

28  Stewart  (Jour,  of  Physiol,  1899  (24),  211)  found  that  in  hemolysis  by 
physical  means  or  under  the  influence  of  serums,  there  is  no  marked  increase  in 
the  electrical  conductivity,  but  hemolysis  by  saponin  and  by  water  causes  an 
increase  of  conductivity,  presumably  because  of  the  escape  of  electrol^'tes ;  cor- 
roborated by  A.  Woelfel,  Biochem.  Jour.,  1908  (3),  146;  see  also  Moore  and  Roaf, 

tuid      D     S  ^ 

"  Rohonyi,  Kolloid-chem.  Beihefte,  1916  (8),  J37,  391.  Knaffel-Lenz  (Arch, 
ges.  Physiol.,  1918  (171),  51)  also  finds  evidence  that  there  is  no  limiting  lipoid 
membrane  about  red  cells. 

30  Kolloid  Zeit.,  1909  (5),  146. 


212  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

sorbed  hemoglobin,  which  process  can  be  dupHcated  experimentally 
with  a  combination  consisting  of  a  corresponding  solid  hydrophilic 
colloid,  fibrin,  and  a  hydrophobic  colloid  dye,  carmin^e;  this  artificial 
combination  behaves  exactly  like  a  corpuscle  to  simple  hemol^'tic 
agents.  ^^ 

Repeated  alternate  freezing  and  thawing  is  another  physical  means 
of  bringing  on  hemolysis.  Heating  to  62°-64°  C.  causes  hemolysis 
of  mammalian  corpuscles;  in  cold-blooded  animals  this  seems  to  occur 
at  a  slightly  lower  temperature.  Hypertonic  solutions  produce  hemo- 
lysis, and  it  may  be  that  freezing  and  desiccating  cause  hemolysis 
through  the  resulting  hypertonicity.^- 

Some  chemical  agents  are  capable  of  liberating  hemoglobin,  even 
when  the  corpuscles  are  in  isotonic  solutions.  The  ordinar}'  salts 
of  serum,  of  course,  do  not  have  this  property,  but  ammonium  salts 
are  strongly  hemolytic.  The  chemical  agents  that  dissolve  red  cor- 
puscles seem  to  be  those  that  have  the  power  of  penetrating  the 
stroma.  Ammonium  salts  and  urea  penetrate  the  corpuscles  freel}^ 
and  cause  hemolysis.  Sugar  and  NaCl  seem  not  to  penetrate  the 
corpuscles,  and  therefore  do  not  produce  hemolysis.  Of  the  perme- 
ating substances,  there  seem  to  be  two  types:  one,  like  urea,  does  not 
produce  hemolysis  when  in  a  solution  of  NaCl  isotonic  with  the  serum; 
the  other,  like  ammonium  chloride,  is  not  prevented  from  producing 
hemolysis  by  the  presence  of  NaCl.^^ 

All  these  agents  seem  to  effect  hemolysis  by  acting  on  the  stroma, 
for  when  the  stroma  of  corpuscles  hardened  in  formalin  has  its  leci- 
thin and  cholesterol  removed  with  ether,  saponin,  a  powerfully  hemo- 
lytic substance,  seems  to  have  no  effect.  The  action  of  saponin  and 
of  many  other  hemolytic  agents  can  be  prevented  b}'  the  presence  of 
cholesterol  in  excess,  suggesting  that  it  is  this  constituent  of  the  stroma 
that  is  affected.^*  By  studying  hemolysis  under  dark  field  illumination 
Dietrich^^  found  that  in  water  hemolysis  a  diffusion  of  hemoglobin 

^^  Concerning  the  influence  of  H-ion  concentration  on  hemolysis  see  Walbum, 
Biochem.  Zeit.,  1914  (63),  221. 

^2  Guthrie,  Jour.  Lab.  Clin.  Med.,  1917  (3),  87. 

^^  Hamburger,  in  his  book,  "Osmotischer  Druck  und  lonenlehre,"  reviews  ex- 
haustively the  physical  chemistry  of  hemolysis.  The  following  is  his  summary 
of  the  permeability  of  red  corpuscles  by  various  substances: 

Organic  Substances. — (a)  Impermeable  for  sugars;  namely,  cane-sugar,  dextrose, 
lactose,  also  arabit  and  mannit.  (6)  Permeable  for  alcohols,  in  inverse  proportion 
to  the  number  of  hydroxyl  groups  that  they  contain;  also  for  aldohj'des  (except 
paraldehyde),  ketones,  ethers,  esters,  antipyrin,  amides,  urea,  urethan,  l)ile  acids 
and  their  salts,  (c)  Slightly  permeable  for  neutral  amino-acids  (glycocoll,  aspar- 
agin,  etc.). 

Inorganic  substances,  not  including  the  salts  of  the  fixed  alkalies,  (a)  Com- 
pletely impermeable  for  the  cations  Ca,  Sr,  Ba,  Mg.  {b)  Pcnncable  for  NHj  ions, 
for  free  acids  and  alkalies. 

^■•Ransom,  Deut.  med.  Woch.,  1901  (27),  194;  Koliert,  "Saponinsubstanzen'' 
Stuttgart,  1904;  Abdcrhalden  and  Le  Count,  Zeit.  exp.  Path.  u.  Ther.,  1905  (2), 
199.  Noguchi  (Univ.  of  Penn.  Med.  Bull.,  1902  (15),  327)  found  lecithin  without 
this  proi)erty. 

"Verb.  Deul.  Patli.  (icscll.,  190S  (12),  202. 


MECHANISM  OF  HEMOLYSIS  213 

takes  place  tluoufih  the  coipusculur  substance,  which  is  not  visibly 
altered;  in  serum  hemolysis  there  is  first  a  precipitate  formed  in  the 
outer  layer,  which  swells.  There  is  no  evidence  that  the  erythrocj^tes 
contain  proteolytic  enzymes  of  their  own  that  might  disintegrate 
them.'*' 

The  fact  that  chloroform,  ether,  bile  salts,  soaps,  and  amjd  alcohol 
will  cause  hiking  is  probably  intimately  connected  with  the  fact  that 
lecithin  and  cholesterol,  important  constituents  of  the  stroma,  are 
both  soluble  in  these  substances.'''  In  general  it  can  be  said  that 
iiemolytic  agents  dissolve  lipoids  or  hydrolyze  proteins  or  lipoids, 
thus  destroying  the  power  of  the  stroma  to  retain  the  hemoglobin.^* 
Nearly  all  the  non-specific  hemolytic  agents  are  inhibited  to  greater 
or  less  degree  by  the  serum,  in  which  inhibition  both  the  proteins  and 
cholesterol  are  concerned. '^^  Cholesterol  also  influences  many  other 
inununity  reactions,  inhibiting  some  and  stimulating  others.*"  The 
resistance  of  the  corpuscles  to  hemolj'sis  by  various  agents  differs 
greatly  in  disease,  although  fairly  constant  in  normal  blood,  the  dif- 
ferences being  caused  in  some  cases  by  changes  in  the  permeability  of 
the  corpuscles,  and  sometimes  by  changes  in  the  environment  of  the 
corpuscle  or  the  presence  of  protective  substances  in  either  the  cor- 
puscles or  the  plasma. 

Arseniuretted  hydrogen,  when  inhaled,  causes  intravascular  hemo- 
lysis, and  there  are  many  other  drugs  and  chemicals  with  the  same 
property,  among  which  may  be  mentioned  nitrobenzol,  nitroglycerin 
and  the  nitrites,  guaiacol,  pyrogallol,  acetanilid,  and  numerous  aniline 
compounds.  Probably  the  hemolysis  produced  by  autolytic  products 
belongs  in  this  category."*^  Alcoholic  extracts  of  tissues  are  com- 
monly hemolytic;  these  extracts  when  added  to  serum  take  on  prop- 
erties which  cause  them  to  resemble  closely  hemolytic  complement 
(Noguchi),  and  the  soaps  seem  to  be  the  active  constituents  of  the 
extracts.  AsHs,  although  strongly  hemolytic  in  the  living  body,  does 
not  hemolyze  corpuscles  in  the  test  tube  (Heffter),  and  this  is  true 
of  some  other  poisons,  which  probablj'  produce  their  effects  through 
tissue  changes. •^^  The  bile  acids  and  their  salts  will  also  produce 
hemolysis,  as  seen  in  jaundice.  Sodium  bicarbonate  solutions  of  one 
or  two  per  cent,  are  hemolytic  for  some  varieties  of  corpuscles,  but 
0.1  per  cent.  Na2C03  and  NaHCOs  do  not  cause  hemolysis.  A  study 
of  the  hemotytic  properties  of  one  class  of  lipolytic  hemolytic  agents, 

38  Von  Roques,  Biochem.  Zeit.,  1914  (64),  1. 

"  See  Koeppe,  Pfiiiger's  Arch.,  1903  (99),  33;  Peskind,  Amer.  Jour.  Phys.,  1904 
(12),  184;  Moore,  Brit.  Med.  Jour.,  1909  (ii),  684. 

38  See  Herzfeld  and  Klinger,  Biochem.  Zeit.,  1918  (87),  36. 

"  See  V.  Eisler,  Zeit.  exp.  Path.,  1906  (3),  296. 

*°  Walbum,  Zeit.  Iminunitiit.,  1910  (7),  544;  Dewey  and  Nuzum,  Jour.  Infect. 
Dis.,  1914  (15),  472. 

"  Concerning  hemolysis  b.y  alcohols,  ketones,  etc.,  organic  acids,  and  essences 
see  Vandevelde,  Bull.  Soc.  chim.  de  Belgique,  1905  (19),  288. 

"  Friedberger  and  Brossa,  Zeit.  Immunitat.,  1912  (15),  506. 


214  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

the  terpenes,  shows  that  their  hemolytic  activity  varies  much  accord- 
ing to  their  physical  properties,  generally  decreasing  directly  with  in- 
crease in  the  solubilitj'  in  water  (Ishizaka).-*^ 

Leucocytes  are  dissolved  by  some  of  these  agents,  particularly  the  bile  salts 
although  they  are  affected  by  no  means  so  rapidh^  or  so  much  as  are  the  erythro- 
cytes. There  seems  to  be  no  relation  between  the  erythroh^tic  and  leucolytic 
powers  of  these  substances.  Water  causes  swelling,  with  solution  of  the  granules 
in  time,  and  the  same  is  true  of  ammonium-chloride  solutions. 

Various  chemicals  cause  morphological  alterations  in  the  leucocytes,  and  of 
bacterial  products  the  toxins  of  pyocyaneus  and  diphtheria  seem  to  be  particularly 
leucocidal,  causing  a  striking  karj'orrhexis  (Schiirmann)." 

Hemolysis  by  Serum 

Normal  blood-serum  of  many  animals  causes  hemolysis  to  greater 
or  less  degree  when  mixed  with  red  corpuscles  of  another  species  of 
animal,  and  this  property  can  be  greatly  increased  by  immunizing 
the  animal  with  red  corpuscles  in  the  usual  way.  This  hemolysis  oc- 
curs both  in  the  test-tube  and  in  the  body,  in  the  latter  case  causing 
severe  anatomical  changes  or  even  death.  In  all  respects  the  mech- 
anism of  hemolysis  by  serum  seems  to  he  identical  with  that  of  bac- 
teriolysis. Two  substances  are  concerned,  one  the  amboceptor,  which 
resists  heat  and  which  is  increased  by  immunizing;*^  the  other,  com- 
plement, which  is  destroyed  at  55°  and  which  is  present  in  normal 
serum.  In  this  case  the  substances  may  be  referred  to  as  hemolytic 
amboceptors  and  hemolytic  complements. 

In  spite  of  the  availability  of  these  particular  cytolytic  substances 
for  study,  very  little  has  been  learned  of  their  exact  nature  and  prop- 
erties. It  is  known  that  amboceptor  is  combined  with  the  red 
cells  in  a  certain  sense  quantitatively,  a  definite  amount  being  re- 
quired to  saturate  a  given  amount  of  corpuscles  so  that  they  will  all 
be  hemolyzed  when  complement  is  added;  and  that  this  reaction  is 
complete  in  less  than  fifteen  minutes  at  45°.  What  change  this  addi- 
tion of  amboceptor  brings  about  in  the  corpuscles  is  unknown.  It 
has  also  been  shown  that  at  0°  the  affinity  between  the  amboceptor 
and  the  corpuscle  is  greater  than  it  is  between  amboceptor  and  com- 
plement, so  that  it  is  possible  at  this  temperature  to  remove  all  the 
amboceptor  from  a  serum  by  treating  it  with  red  corpuscles,  and 
thus  we  can  obtain  complement  free  from  amboceptor.  This  experi- 
ment also  shows  that  the  two  bodies  exist  side  by  side  in  the  serum 
without  combining,  and  that  combination  occurs  only  after  the  ambo- 
ceptor has  become  united  to  the  erythrocyte.  Moreover  the  hemoly- 
tic amboceptor  can  be  separated  from  the  antigen  to  which  it  has  been 

"  Arch.  exp.  Path.,  1914  (75),  195. 

**  Cent.  f.  Pathol.,  1910  (21),  337. 

*^  In  an  extensive  study  of  the  hemolytic  antiI)ody,  Thiele  and  Embleton 
(Zeit.  Imnuinitiit.,  1913  (20),  1)  describe  its  formation  as  in  several  steps,  at  first 
being  tlicrmolabile  and  uniting  with  the  corpuscle  only  when  warmed.  They 
also  find  complt^ment  to  have  sev(>ral  components.  This  is  not  coufiriued  by  Siier- 
man,  Jour.  Infect.  Dis.,  1918  (22),  534. 


HEMOLYTIC  AMBOCEl'TOR  215 

combined.'"'  Hemolysis  hy  iimmine  sera  takes  place  l)est  in  a  medium 
with  a  reaction  corresponding  to  that  of  the  blood,  acids  being  more 
iiarmful  than  alkalies;  with  unfavorable  reaction  the  complement  does 
not  unite  with  the  aml^oceptor,  although  the  latter  unites  with  the 
corpuscle.'^ 

The  Amboceptor. — Amboceptor  is,  a.s  a  rule,  destroyed  hy  heating  to  70"  or 
liifiiher.'^  Its  place  of  origin  is  unknown.  Metchnikolt  holds  that  it  is  derived 
chiefly  from  the  leucocytes,  in  support  of  which  view  is  the  fact  that  leucocytes 
dissolve  red  corpuscles  after  ingesting  them;  however,  other  phagocytic  cells 
have  the  same  power,  particularly  endothelial  cells,  and  it  is  an  open  question 
whether  the  intracellular  digestion  of  engulfed  cells  is  the  same  process  as  extracel- 
lular hemolysis;  prol)ably  it  is  not,  for  there  seem  to  be  more  disintegrative  changes 
in  intracellular  digestion  than  in  hemolysis.  Quinan^^  found  that  the  diffusible 
constituents  of  hemolytic  serum  played  no  role  beyond  that  of  maintaining  os- 
motic pressure.  He  was  unable,  however,  to  localize  the  immune  body  in  any  of 
the  protein  constituents,  and  Liebermann  and  Fenyves.sy^"  believe  that  they 
obtained  the  amboceptor  in  a  protein-free  condition,  in  which  it  behaves  like 
a  weak  acid.  Amboceptors  are  insoluble  in  lipoids  or  lipoid  solvents  (Meyer),*' 
and  they  move  towards  the  cathode  in  an  electric  field,  as  do  other  antibodies. *- 
The  amboceptor  complement  reaction  resembles  a  bimolecular  reaction  wliich  is 
accelerated  bj'  its  end  products  (v.  Krogh).*'  Many  of  the  effects  of  hemolytic 
amboceptors  can  be  duplicated  with  silicic  acid;^^  and  a  dye,  brilliant  green,  may 
in  minute  quantities  sensitize  corpuscles  so  that  they  are  hemolyzed  by  very 
small  amounts  of  normal  serum,  or  by  lecithin.^^ 

The  amboceptors  of  normally  hemolytic  serum  seem  to  be  no  different  from 
those  in  immune  serum,  and  amboceptors  of  one  animal  can  combine  with  comple- 
ment furnished  by  the  serum  of  an  entirely  different  animal.  It  is  the  amboceptor 
alone  that  gives  the  specific  nature  to  the  reaction,  and,  as  is  the  case  with  all 
other  immunizations,  it  is  very  difficult  to  secure  antibodies  by  immunizing  an 
animal  with  blood  from  another  animal  of  its  own  species,  isohemolysiris.  The 
place  of  origin  of  hemolysins  is  unknown,  as  with  other  antibodies,  but  that  it  is 
not  in  the  blood  seems  to  have  been  established  conclusively  by  Hektoen  and 
Carlson.**  Immune  hemolysins  cannot  pass  from  the  mother  to  the  fetus  before 
birth*"  but  they  can  be  transmitted  through  the  colostrum  (Famulener).*^ 

Although  Ehrlich  held  that  the  union  between  cell  and  amboceptor  is  pureh' 
chemical  and  follows  ordinary  chemical  laws,  especially  the  law  of  multiple  pro- 
portions, Bordet  and  other  French  observers  have  claimed  that  the  union  between 
amboceptor  and  corpuscle  is  physical  and  not  chemical.*^     Probably  the  union  is 

*''  Kosakai,  Jour.  Immunol.,  1918  (3),  109. 

*''  Michaehs  and  Skwirsk}',  Zeit.  Immunitat.,  1909  (4),  357. 

^^  Ultraviolet  light  destroys  immune  hemolysin  (Stines  and  Abelin,  Zeit. 
Immunitat.,  1914  (20),  598). 

"  Hofmeister's  Beitr.,  1904  (5),  95. 

*°  Jahresber.  d.  Immunitat.,  1911  (7),  2. 

*'  Ibid.,  1909,  Vol.  3. 

"  Teague  and  Buxton,  Jour.  Exper.  Med.,  1907  (9),  254. 

"  Biochem.  Zeit.,  1909  (22),  132. 

"  Landsteiner  and  Rock,  Zeit.  Immunitat.,  1912  (14),  14. 

**  Browning  and  Mackie,  Zeit.  Immunitat.,  1914  (21),  422. 

*«  Jour.  Infect.  Dis.,  1910  (7),  319. 

"  See  Sherman  concerning  normal  antibodies  in  the  fetus.  Ibid.,  1918  (22),  534). 

"76id,  1912  (10),  332. 

^'  Bang  and  Forssmann  (Hofmeister's  Beitr.,  1906  (8),  238)  suggest  that  the 
amboceptor  merely  renders  the  corpuscle  permeable  for  the  complement,  perhaps 
through  action  on  the  lipoid  membrane;  the  complement  then  acts  directly  upon 
some  constituent  of  the  corpuscle,  without  the  amboceptor  acting  as  a  combining 
substance  in  any  way.  They  found  that  the  substance  in  blood  which  stimulates 
the  antibody  formation  in  the  case  of  hemolysin  formation,  is  chemically  separable 
from  the  substance  in  blood  which  unites  with  these  antibodies;  therefore,  they 
conclude,  the  "receptors"  of  cells  are  not  identical  with  the  antibodies.  (See  con- 
troversy with  Ehrlich  in  IMiinch.  nied.  Woch.,  "N'ols.  56  and  57.) 


216  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

with  the  stroma  rather  than  with  the  hemoglobin,  and  the  result  of  the  union  is  to 
render  the  stroma  permeable  to  the  hemoglobin,  or  to  separate  the  bonds  that 
unite  the  hemoglobin  to  the  stroma,  f^"  There  are  grounds  for  believing  that  the 
amboceptor  not  only  binds  the  complement,  but  that  it  also  produces  changes  in 
the  corpuscles  (Muir).  Mathes^*  contends  that  red  corpuscles  cannot  be  dissolved 
by  hemolytic  serum  or  by  pancreatic  juice  until  after  they  have  been  killed;  as 
heated  serum  does  not  kill  them,  this  is  presumably  done  by  the  complement. 
Corpuscles  that  have  been  killed  can  then  be  dissolved  in  their  own  serum.  Le- 
vene^^  tried  to  produce  hemolytic  serums  by  immunizing  with  different  consti- 
tuents of  corpuscles,  using — (1)  pure  crystalline  hemoglobin;  (2)  proteins  of  the 
stroma  soluble  in  salt  solutions;  (3)  an  extract  with  alcohol-ether;  and  (4)  an  extract 
in  1.5  per  cent,  sodium  bicarbonate.  Only  the  last  gave  positive  results,  and  the 
serum  was  almost  devoid  of  agglutinative  properties.  Injection  with  corpuscles 
that  had  been  digested  with  trypsin  gave  about  the  same  results  as  alkaline  ex- 
tracts; corpuscles  digested  by  pepsin  gave  a  much  weaker  serum;  in  neither  was 
agglutination  obtained.  According  to  Bang  and  Forssmann*'^  and  others  ethereal 
extracts  of  red  corpuscles  give  rise  to  production  of  hemolysins  on  immunization, 
and  this  "lysinogen"  substance  can  be  precipitated  with  acetone,  is  insoluble 
in  alcohol,  is  not  destroyed  by  boiling,  and  gives  rise  to  no  agglutinin.  Numerous 
other  olDservers,  however,  have  failed  to  confirm  these  findings.  Ford  and  Halsey^* 
ol)tained  serum  with  both  lytic  and  agglutinative  powers  by  injecting  either  the 
stroma  or  the  laked  blood  free  of  stroma.  Stewart^^  obtained  similar  results  by 
immunizing  with  corpuscles  laked  by  physical  means,  by  serums,  or  by  saponin. 
Pure  hemoglobin  itself  is  not  antigenic.  ^^  According  to  Guerrini,*^^  nucleoprotein 
obtained  from  dog's  blood  engenders  specific  hemolysins,  and  Beebe  states  that 
nucleoproteins  from  visceral  organs  do  not  have  this  effect.  Levene's  alkaline 
erythrocyte  extracts  probably  also  contained  nucleoproteins.  Vedder,^*  was  unable 
to  produce  hemolysins  when  he  used  ether  extracts  of  corpuscles  as  antigen,  or  with 
globulin  from  stroma,  but  the  protein  extract  left  after  removing  the  globulin, 
presumably  albumin,  as  well  as  lipoid-free  stroma,  produced  hemolj-sin.  The 
henolysin  itself  seems  to  be  a  globulin.  On  the  other  hand,  Bennett  and  Schmidt^' 
obtained  hemolysin  by  immunizing  with  the  globulin  precipitated  from  hemolyzed 
erythrocytes  by  CO2. 

Immunization  with  extracts  of  tissues  and  cells  of  various  sorts,  even  when 
entirely  free  from  blood  (e.  g.,  spermatozoa),  may  produce  hemolytic  sera.  The 
fact  that  various  tissues  from  many  different  species  of  animals,  when  used  as 
antigen,  may  give  rise  to  hemolysin  for  sheep  corpuscles,  is  an  interesting  but  so 
far  unexplained  phenomenon,  which  is  discussed  under  "Specificity"  (Chapter 
vii). 

The  Complement. — Hemolytic  complement  possesses  the  same  properties  as 
bacteriolytic  complement,  resembling  enzymes  to  the  extent  that  it  is  susceptible 
to  heat,  causes  a  disintegration  of  cells,  and  is  largely  retained  by  Berkefeld 
filters.'"  The  joint  action  of  amboceptor  and  complement  is  strikingly  like  the 
activation  of  trypsinogen  by  kinase.  On  the  other  hand,  hemolysis  by  serum  is 
quite  different  from  the  effect  of  trypsin  on  corpuscles,  as  trj-psin  completely  dis- 
organizes the  hemoglobin  and  destroys  the  stroma,  while  in  hemolysis  the  stroma 
and  hemoglobin  seem  to  be  merely  separated  from  one  another  but  not  chemically 
altered.     Again,    hemolysin  acts  quantitatively,  although  that  may  be  due  to  a 

^"  Corpuscles  treated  with  osmic  acid  will  unite  with  hemolysins  of  diverse 
origin,  but  when  used  for  immunizing  they  engender  no  hemolysins  (Coca;  also 
V.  Szily,  Zeit.  Immunitiit.,  1909  (3),  451).  Heating  corpuscle  stroma  alters 
greatly  the  reactivity  (Landstciner  and  Frasek,  ibid.,  1912  (13),  403). 

"  iVIiinch.  med.  Woch.,  1902  (49),  8. 

"  Jour.  Med.  Research,  1904  (12),  191. 

"  Hofmeister's  Beitr.,  1906  (8)  238. 

"  Jour.  Med.  Research.,  1904  (11),  403. 

^'  Amer.  Jour,  of  Physiol.,  1904  (11),  250. 

8"  .Schmidt  and  liennctt,  Jour.  Infect.  Dis.,  1919-(25),  207. 

"  Riv.  crit.  di  clin.  mod.,  1903  (4),  561. 

"8  Jour.  Immunol.,  1919  (4),  141. 

"  Jour.  Immunol.,  1919  (4),  29. 

'0  Muir  and  Browning,  Jour.  Path,  and  Bact.,  1909  (13),  232. 


HEMOLYTIC  COMPLEMENT  217 

difFercnce  in  tlie  waj'  the  biiidinp;  to  the  coll  occurs,  rather  than  in  the  nieth(Ki  of 
action  of  the  complement.  Landsteiner  and  others  have  suRKested  that  a  lij)oir|al 
complement  dissolves  the  corpuscle  lipoids,  lil)eratinK  the  hemoRlohin,  while 
Neiiher^  and  others  have  supported  the  hypothesis  that  conipieiuent  is  virtually 
a  lipase  which  splits  the  lipnids  out  of  the  corj)Uscles.  Hordct  believes  that  the 
hemolysin  causes  a  lesion  of  the  stroma  which  chanses  the  resistance  to  osmotic 
influences.  Complement  is  present  in  the  plasma  in  about  the  same  amounts 
as  in  the  corresponding  sera,  so  it  is  not  a  substance  set  free  only  by  coaf^uiation  of 
the  blood  (W'atanabe).'"  Dick'^  has  found  evidence  that  the  complement  is  a 
ferment  formed  in  the  liver,  and  that  it  causes  actual  proteol5'tic  chanp;es.  Job- 
ling"  associates  the  serum  lipase  with  the  hemolytic  complement.'*  Ohta'* 
ol)served  no  increase  in  non-coagulable  nitrogen  during  hemolysis,  but  Dick 
found  an  increase  in  the  free  amino  acids;  therefore,  as  yet  agreement  has  not  been 
reached  as  to  whether  hemolysis  depends  in  any  way  upon  proteolysis  or  lipolysis 
in  the  corpuscle  stroma. 

Although  the  serum  of  one  animal  may  complement  the  immune  bodies  in 
serum  of  several  other  varieties,  and  also  produce  lysis  of  many  sorts  of  cells,  it  may 
be  that  not  one  complement  does  all  the  complementing;  Ehrlich  and  others  have 
asserted  that  one  serum  may  contain  several  complements  of  slightly  differing 
natures.  Noguchi,'"  Liebermann  and  Fenyvessy,  and  others  have  pointed  out  the 
striking  resemblance  between  hemolytic  complement  and  certain  compounds  of 
soaps  or  lipoids  with  serum  proteins,  and  it  is  possible  that  such  compounds  are 
of  importance  in  serum  hemolysis;  but  there  seems  also  to  be  evidence  of  the 
existence  of  distinct  protein  complements,  entirely  different  from  these,"'  and  it  is 
possible  that  the  protein  complements  are  the  important  agents  in  specific  hemo- 
lysis by  immune  sera.'* 

Antibodies  can  be  obtained  for  both  complement  and  hemolytic  amboceptor  by 
immunizing  against  serum  containing  them,  and  in  manj^  serums  antihemolysins 
exist  normally.  Against  certain  vegetable  hemolysins  this  antihemolytic  action  is 
very  strong  (Kobert).  Antihemolysins  are  generally  anticomplements,  but  in  a 
number  of  instances  anti-amboceptors  have  been  obtained.  The  existence  of  im- 
mune bodies  specific  for  hemolytic  amboceptor  and  complement,  supports  the  view 
that  both  of  these  agents  are  proteins. 

In  hemolysis  as  in  bacteriolysis  the  complement  exhibits  two  function^,  corres- 
ponding to  the  "end-piece"  and"  mid-piece"  fractions.  Herzfeld  and  Klinger'^o  con- 
sider the  mid-piece  to  be  a  globulin  which  renders  the  surface  of  the  corpuscles  more 

"'  Jour.  Immunol.,  1919  (4),  77. 

"2  Jour.  Infect.  Dis.,  1913  (12),  111. 

"  Jobling  and  Bull,  Jour.  Exper.  Med.,  1913  (17),  61;  also  Bergel,  Deut.  Arch, 
klin.  Med.,  1912  (106),  47. 

~*  Thiele  and  Embleton,  however,  state  that  hemolysin  is  not  a  lipase,  and 
that  the  hemolytic  power  of  serum  has  no  relation  to  its  lipolytic  power  (Jour. 
Path,  and  Bact.,  1914  (19),  349). 

"  Biochem.  Zeit.,  1912  (46),  247;  see  also  McNeil  and  Kahn,  Jour.  Immunol., 
1918  (3),  295. 

'«  Biochem.  Zeit.,  1907  (6),  172  and  327;  Jour.  Exper.  Med.,  1907  (9),  436. 

''  SeeLiefmann,  el  al.,  Zeit.  Immunitiit.,  1912  (13),  150. 

^'Liebermann  and  Fenyvessy  (loc  cit.y""  believe  that  serum  hemolysis  takes 
place  as  follows:  First,  the  amboceptor  acts  on  the  corpuscle,  injuring  it  so 
that  it  becomes  less  resistant;  second,  this  combination  acts  upon  the  comple- 
ment (a  soap  compound)  and  frees  the  soap  so  that  it  can  unite  with  the  ambo- 
ceptor-corpuscle  system;  third,  the  soap  causes  hemolysis;  fourth  (as  a  separate 
step),  the  escape  of  the  hemoglobin  from  the  corpuscles.  Tissot  ascribes  import- 
ance to  the  fatty  acids  of  the  plasma  (Compt.  Rend.  Acad.  Sci.,  1919  (168),  1283). 
Bergel  (Zeit  Immunitiit.,  1918  (27),  441)  supports  the  hypothesis  that  immune 
hemolysis  and  agglutination  depend  on  a  solution  of  the  lipoids  of  the  cells.  In 
this  reaction  the  lipoids  act  as  antigen,  the  new-formed  amboceptor  is  formed  by 
the  lipoids  of  the  lymphocj-tes  as  a  zymogen  which  is  activated  by  serum  comple- 
ment, and  is  specifically  bound  by  the  lipoid  antigens  of  the  corpuscles.  That  is, 
the  lipoids  are  the  haptophore  groups  of  the  antigen;  they  bind  the  receptor  of  the 
thermostabile  lipase  zymogen,  which  is  activated  by  the  non-specific  complement. 

'8"  Biochem.  Zeit.,  1918  (87),  36. 


218  CHEMISTRY  OF   THE  IMMUNITY  REACTIONS 

capable  of  taking  up  certain  disintegration  products  contained  in  the  serum  (per- 
sensitization)  which  constitute  the  so-called  end-piece,  and  which  produce  hemo- 
lysis by  direct  hydrolysis  or  solution  of  the  stroma  elements. 

Hemagglutinin. — Agglutination  of  red  corpuscles  occurs  under 
the  influence  of  immune  serum  as  well  as  under  the  influence  of  some 
normal  serums.  In  all  respects  the  principles  seem  to  be  the  same  as 
those  described  for  bacterial  agglutination.  The  hemagglutinating 
antibody  behaves  like  the  other  antibodies  and  proteins  under  the  in- 
fluence of  chemical  and  physical  agencies,  but  Landstciner  and  Jagic 
have  obtained  strong  agglutinating  solutions  containing  very  little 
protein.  BergeF^  contends  that  hemagglutination  is  produced  by 
lipase  from  the  lymphocytes,  which  alters  the  lipoid  membranes  of 
the  erythrocytes.  Agglutination  occurs  at  much  lower  temperatures 
than  hemolysis,  and  also  is  not  checked  by  heating  the  serum  to  55° ; 
hence  it  is  possible  to  observe  hemagglutination  independent  of  hemo- 
lysis. Serums  may  contain  hemagglutinins  and  not  be  hemolytic; 
the'reverse  is  also  true.  The  conglutmin  effect  of  beef  serum  (Bordet 
and  Gay)  is  also  observed  with  corpuscles  as  with  bacteria.  As  agglu- 
tination occurs  in  corpuscles  that  have  been  fixed  in  formalin  or  sub- 
limate' it  is  probably  not  the  proteins  that  are  affected,  but  some  other 
of  the  ingredients  of  the  stroma,  of  which  lecithin  and  cholesterol  seem 
to  be  the  chief. 

Certain  vegetable  poisons  produce  agglutination  of  red  corpuscles, 
especially  ricin,  abrin,  and  crotin,  and  the  fact  that  ricin  has  httle 
or  no  hemolytic  action  shows  the  independence  of  the  processes.  Anti- 
sera  for  these  vegetable  poisons  are  also  antiaggiutinative,  acting,  as 
Ehrlich  showed,  on  the  poison  and  not  on  the  corpuscles.  The  seeds 
of  many  non-poisonous  leguminous  plants,  and  also  of  Solanaceoe, 
yield  extracts  that  are  strongly  agglutinative  for  red  corpuscles;  in 
Phaseolus  multiflorus  the  active  substance  is  found  in  the  proteose 
of  the  seed,  and  seems  to  be  a  part  of  the  stored  food  (Schneider).^" 
It  is  not  present  in  other  parts  of  the  plant.  Snake  venojus  contain 
agglutinins,  destroyed  by  heating  to  75°;  their  agglutinating  power 
being  in  inverse  ratio  to  their  hemolytic  power.  Corpuscles  aggluti- 
nated by  venoms  may  be  again  separated  by  potassium  permanganate 
solutions. ^^  Silicic  acid  and  certain  other  colloids  may  act  as  agglu- 
tinins, their  effects  bearing  a  relation  to  the  effects  of  electrical  charges 
upon  agglutination  of  bacteria  or  of  colloids  {q.  v.).^^  Corpuscles 
that  have  been  sensitized  by  hemolytic  amboceptors  are  much  more 
readily  agglutinated  by  salts  of  heavy  metals,  especially  copper  and 
zinc,  presumably  because  of  quantitative  alterations  in  the  electrical 
charge  of  the  corpuscles  induced  by  the  antibody.'** 

"  Zoit.     Iininunitat.,  1912  (14),  255;  1913  (17),  109. 

8»,J()ur.  liiol.  Chom.,  1912  (11),  47;  l)il)li()grapliv. 

»'  .See  FlexntT,  irniv.  of  Pmn.  Mod.  Bull.,  1902  (15),  .T24  and  :J»)1. 

82  S(>e  J/indstrinor  aiul  .JaKJc.  Aliiiich.  nicd.  \\'ocli.,  1901  (51),  1185. 

83  lOisiicr  and  T'lirdcinaiin,  Zcit.  Iiuniuiiitat.,  1914  (21),  520. 


HEMOLYSIS  HY  JiACTKRIA  210 

Agglutination  of  the  corpuscles  during  life  may  bo  of  great  patho- 
logical importance,  for  such  masses  of  agglutinated  corpuscles  may 
readily  produce  capillary  thrombi  and  emboli,  which,  if  wide-spread, 
may  create  much  disturbance.  Somethmes  the  serum  of  one  indi- 
vidual of  a  species  agglutinates  the  corpuscles  of  another  individual 
of  the  same  species  (isoagglutination) j^^"  a  fact  which  must  be  taken 
into  account  in  performing  transfusion  of  blood,  lest  dangerous  ag- 
glutination take  place.  Agglutination  of  an  individual's  corpuscles 
by  his  own  serum  {autoagglutination) ,  may  also  be  observed  under 
experimental,  and  perhaps  under  pathological  conditions  (Land- 
steiner),**^  this  pathological  autoagglutination  probably  occurring 
especially  at  temperatures  below  37°.  (See  Paroxysmal  Hemoglobin- 
uria.) Many  bacteria  produce  substances  that  are  agglutinative  for 
human  red  corpuscles,  among  them  being  B.  typhosus,  pyocyaneus, 
and  staphylococcus.  Flexner^^  has  found  in  typhoid  fever  thrombi 
that  seemed  to  be  composed  of  agglutinated  red  corpuscles,  almost 
free  from  fibrin  and  leucocytes.  Probably  many  of  the  so-called 
"hyaline  thrombi"  found  frequently  in  infectious  diseases  are  really 
composed  of  agglutinated,  partly  hemolyzed  red  corpuscles  (see 
"Thrombosis,"  Chap.  xiii). 

Hemolysis  by  Bacteria^^ 

Both  pathogenic  and  non-pathogenic  bacteria  produce  hemolytic 
substances  that  are  excreted  into  the  fluids  in  which  they  grow.  Dur- 
ing many  infectious  diseases  marked  hemolysis  occurs,  especially  in 
those  diseases  accompanied  by  septicemia.  After  death  the  hemo- 
globin of  the  blood  goes  into  solution,  and  the  resulting  staining  of 
the  walls  of  the  blood-vessels,  and  later  of  the  tissues  everywhere,  is 
generally  familiar.  In  the  post-mortem  hemolysis  probably  the  pu- 
trefactive organisms  are  chiefly  concerned,  although  it  is  marked  a 
very  short  time  after  death  in  many  cases  of  septicemia,  particularly 
when  the  infecting  organism  is  the  streptococcus,  and  here  probably 
the  pathogenic  organism  is  the  chief  cause  of  the  hemolysis.  The 
hemolytic  action  of  bacteria  can  be  studied  both  in  vitro  and  in  vivo. 
Among  the  best  known  hemolytic  bacterial  toxins  are  tetanolysin, 
pyocyanolysin,  typholysin,  staphylolysin,^''  and  streptocolysin,  as  they 
have  been  termed.     Of  tliese,  the  case  of  pyocyanolysin  is  question- 

"«  Review  bv  Happ,  Jour.  Exp.  Med.,  1920  (31)  313. 

s^  See  also  Clough  and  Richter,  Bull.  Johns  Hop.  Hosp.,  1918  (29),  86;  Rous 
and  Robertson,  Jour.  Exp.  Med.,  1917  (27), 509. 

"  Univ.  of  Penn.  Med.  Bull.,  1902  (15),  324;  Amer.  Jour.  Med.  Sci.,  1903  (126), 
202. 

*^  See  Pribram,  Kolle  and  Wassermann's  Handbuch.,  1913  (II),  1328. 

*"  Analysis  of  staphj-loh^sin  by  Burkhardt  (Arch.  exp.  Path,  und  Pharm.,  1910 
(63),  107),  showed  it  to  be  dialyzable,  protein-  and  biuret-free,  thermolabile  and 
soluble  in  ether.  From  B.  puiidum  he  isolated  a  hemolj'tic  substance  which 
seems  to  be  a  derivative  bj'  oxidation  of  erucacic  acid  (oxj-diniethylthiolerucacic 
acid). 


220  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

able,  because  it  has  been  described  as  resisting  heat  above  the  boil- 
ing-point, and  Jordan'^^  seems  to  have  proved  that  the  hemolysis  is 
ascribable  to  the  alkalinity  that  this  organism  produces  in  culture- 
media.  Other  bacterial  hemolysins  are,  however,  destroyed  by  heat 
at  70°  or  less  for  two  hours;  but  they  are  altogether  different  from 
ordinary  immune  hemolysins.  Apparently  streptocolysin  is  simph'  a 
toxin  for  red  cel,ls,^^  and  unites  directly  to  the  cell  receptois  without 
the  intervention  of  any  intermediary  body.  As  a  similar  structure  has 
been  shown  for  staphylolysin  and  tetanolysin,  it  is  probable  that  the 
bacterial  hemolysins  are  all  merely  toxins  with  a  particular  affinity  for 
red  cells,  and  against  some  of  these  bacterial  hemotoxins  antitoxic  sera 
are  obtainable,  although  there  is  usually  some  question  as  to  how  much 
of  the  antagonistic  effect  depends  on  true  antitoxins  and  how  much 
upon  the  cholesterol  in  the  serum.  However,  a-  strong  antiserum  has 
been  obtained  against  the  hemotoxin  of  B.  Welchii.^^  Of  course 
bacteria  may  also  form  many  non-specific  hemolytic  substances  as 
products  of  their  metabolism,  such  as  acids  and  bases. 

Secondary  anemia  occurring  in  the  infectious  diseases  is  probably 
to  be  explained  largely  by  this  hemol3^tic  property  of  bacterial  toxins. 
Hemoglobinuria  may  also  be  produced  in  the  same  way  in  some  in- 
stances. Intravenous  injections  of  filtrates  of  the  saprophyte,  B. 
megatherium,  will  produce  hemoglobinuria  in  guinea-pigs,  hence  hemo- 
lysis is  not  an  exclusive  property  of  pathogenic  bacteria,  and  with 
streptococci  LyalP^  found  that  the  hemolysin  titer  did  not  afford  a 
criterion  of  virulence.  No  immunity  to  streptococci  is  produced  in 
animals  immunized  with  streptococcus  hemolysin.^-  Pneumococci 
produce  an  intracellular  hemolytic  toxin  which  is  very  labile  and 
antigenic;  living  pneumococci  convert  hemoglobin  into  methemoglobin, 
but  this  the  hemolytic  extracts  of  pneumococci  cannot  do  (Cole).^^ 
Streptococcus  viridans  has  the  same  propertj^,^*  which  may  play  a 
part  in  the  effects  of  infections  with  these  organisms,  von  Hellens^* 
states  that  streptocolysin  is  ether  soluble  and  heat  resistant. 

Hemolysis  by  Vegetable  Poisons 

A  number  of  plant  poisons  are  strongly  homolj'tic,  and  some  of 
them  owe  much  of  their  to.xicity  to  their  effect  on  the  erj'throcytes. 
One  group  consists  of  the  bodies  often  called  "vegetable  toxalbu- 
mins,"  because  they  seem  to  be  proteins,  and  includes  ricin,  abrin, 
crotin,  curcin  and  robin. ^^     Of  these,  crotin  and  curcin  are  particu- 

«8  Jour.  Medical  Research,  1903  (10),  31. 

«9  Jour.  Amer.  Med.  Assoc,  1903  (41),  962;  Jour.  Infect.  Dis.,  1907  (4),  277. 
s"  Ford  and  Williams,  Jour.  Immunol.,  1919  (4),  385. 
»'  Jour.  Med.  Kes.,  1914  (30),  515. 

»2  McLcod  and  McXeo,  .lour.  Path,  and  Bact.,  1913  (17),  524. 
»3  Jour.  J']xper.  Med.,  1914  (20),  347,  3ti3. 
»'  liiakc,  Jour.  Expcr.  Mod.,  1910  (24),  315. 
.     »"  Cent.  f.  Bakt.,  1913  ((iS),  002. 

^^  The  sap  of  (Cotyledon  Schcidcckeri  contains  hemolytic  substances  of  peculiar 
character.     (See  Kritchevvski,  Jour.  Exp.  Med.,  1917  (20),  069.) 


HEMOLYSIS  BY  SAPONINS  221 

larly  actively  licuiolytic,  while  riein,  abiin,  and  robin  are  more  marked 
by  their  agglutinating  action,  hemolysis  being  produced  only  by 
relatively  large  doses.  Their  effects  vary  greatly,  however,  according 
to  the  species  of  animals  whose  blood  is  used.  They  resemljle  the 
bacterial  toxins,  in  that  immunity  can  be  secured  against  them,  and 
the  immune  serum  will  prevent  their  hemolytic  action.  Heating 
the  toxalbumins  to  65°  or  70°  does  not  destroy  the  hemolytic  or 
agglutinating  action  except  with  phallin,  but  100°  does.  The  action 
of  these  substances  is  not  like  that  of  the  enzymes,  in  that  it  is 
quantitative,  a  given  amount  acting  on  a  given  amount  of  cor- 
puscles to  which  it  is  bound.  Madsen  and  Walbum"  observed  that 
red  corpuscles  had  the  power  of  dissociating  neutral  mixtures  of 
ricin  and  antiricin,  the  ricin  entering  the  corpuscles  from  which  it 
could  be  recovered. ^'^  Ford  and  Abel  believe  the  hemolytic  agent  of 
amanita  to  be  a  glucoside.  (The  general  nature  and  other  properties 
of  these  substances  are  considered  under  the  heading  of  "  Phytotoxins," 
in  Chap,  vi.) 

Saponin  Group. — Another  quite  distinct  group  of  vegetable 
hemolyzing  agents  consists  of  the  "saponin  substances. "^^  These 
are  a  closely  related  group  of  glucosides,  found  in  at  least  46  differ- 
ent families  of  plants,  and  they  are  strong  protoplasmic  as  well  as 
hemolytic  poisons.  They  differ  altogether  from  the  true  toxins,  be- 
ing heat  resistant,  having  no  resemblance  to  proteins,  and  not  giving 
rise  to  antibodies  on  immunization  of  animals.^  The  degree  of  their 
toxicity  is  not  directly  proportional  to  their  hemolytic  activity;  they 
seem  to  injure  chiefly  the  nerve-cells.  Apparently  hemolysis  is 
brought  about  by  action  upon  the  lipoids  of  the  red  corpuscles,  for 
addition  of  cholesterol  to  saponin  prevents  its  hemolytic  effect;^  leci- 
thin does  not  have  the  same  property.^  Both  cholesterol  and  leci- 
thin combine  with  saponin,  the  cholesterol  compound  being  quite 
inert,  whereas  the  lecithin  compound  is  both  hemolytic  and  toxic. 
The  compound  formed  between  a  typical  saponin,  digitonin,  and 
cholesterol,  is  so  insoluble  that  it  has  been  found  useful  in  the  quan- 
titative analysis  of  cholesterol.'*     Normal  serum  seems  to  contain 

"  Cent.  f.  Bakt.,  1904  (36),  242. 

^^  According  to  Pascucci  (Hofmeister's  Beitr.,  1905  (7),  457),  ricin  combines 
directly  with  lecithin,  the  compound  being  strongly  hemolytic. 

^^  Complete  literature  on  saponin  given  by  Kobert,  "Die  Saponinsubstanzen," 
Stuttgart,  1904;  also  Kunkel,  "Handbuch  der  Toxokologie,"  Jena. 

1  Saponins  are  characterized  by  their  ready  solubility  in  water  and  the  foam- 
ing, soapy  character  possessed  by  the  solution;  hence  their  technical  applications 
as  soap  bark,  etc.  Heated  with  dilute  acids  they  split  off  sugar;  also  when  acted 
on  by  glucoside-splitting  enzymes  (from  spiders),  according  to  Kobert.  Saponin 
from  Quillaja  (soap-bark)  has  the  formula  C19H30O10  (Stiitz).  Most  are  colloids, 
but  some  crystallize. 

2  Ransom,  Deut.  med.  Woch.,  1901  (27),  194;  Madsen  and  Xoguchi,  Cent.  f. 
Bakt.,  1905  (37),  367;  Pascucci,  Hofmeister's  Beitr.,  1905  (6),  543. 

3  Noguchi,  Univ.  of  Penn.  Med.  Bull.,  1902  (15),  327;  Meyer,  Hofmeister's 
Beitr.,  1908  (11),  357. 

^  Windaus,  Chem.  Berichto,  1909  (42),  238. 


222  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

an  antihcmolysin  for  saponin,  and  therefore  hemoglobinuria  is  not 
produced  by  all  saponins  on  intravenous  injection.  Careful  immu- 
nization leads  to  a  slight  increase  in  this  antihemolytic  action  of  the 
serum,  possibly  due  to  an  increased  formation  of  cholesterol  (Ko- 
bert).  The  resistance  of  corpuscles  to  saponin  hemolysis  varies  in 
disease,  being  especially  low  in  jaundice  (M'Neil).^ 

A  study  of  the  toxicity  of  the  members  of  this  group  by  Kobert^ 
shows  that  in  general  they  have  similar  properties,  but  that  minor 
differences  exist  between  them.  All  cause  hemolysis,  some  in  dilu- 
tion as  great  as  1:100,000.  Some  produce  hemoglobinuria  when  in- 
jected intravenously,  others  do  not.  All  paral3^ze  the  heart,  but  the 
injuries  to  the  central  nervous  system  are  the  chief  cause  of  death. 
Marked  local  changes  are  produced  at  the  site  of  injection,  but  the 
leucocytes  are  apparently  not  injured,  although  sterile  suppuration 
is  produced.  There  is  a  period  of  latency  after  intravenous  injection 
of  small  doses — twenty-four  hours  or  more — before  the  tappearance 
of  symptoms. 

Sapotoxin  is  one  of  the  most  actively  toxic  and  hemolytic  products  of  quillaja. 

Cyclamin  is  also  a  member  of  this  group  (derived  from  Cyclamen),  and  is 
said  to  be  the  most  active  of  all  as  a  hemolytic  agent  (Tufanow). 

SoLANiN^  is  obtained  from  all  parts  of  the  potato  plant,  combined  with  malic 
acid;  it  is  found  particularly  in  young  sjjrouts,  but  not  in  any  considerable  amounts 
in  normal  potatoes.^  Its  formula  is  unknown  ,but  as  it  splits  up  into  an  alkaloid 
{solanidin)  and  sugar  it  is  called  a  gly co-alkaloid.  In  its  action  it  resembles  the 
saponins,  being  a  powerful  protoplasmic  poison,  killing  bacteria,  and  hemolyzing 
blood  in  very  great  dilutions." 

A  great  number  of  hemolytic  poisons  are  obtained  from  poisonous  mushrooms. 
Best  known  of  these  is: 

Helvellic  Acid,  from  Helvella  esculenta,  which  has  the  empiric  formula 
CiaHiiiO?.'"  Intravenously  injected  it  produces  hemoglobinuria  and  icterus,  with 
hemoglobin  infarcts  in  the  kidneys  (Bostrocm)." 

Phallin,  or  Amanita  hemolysin,  described  by  Robert  as  a  toxalbumin,  has 
been  found  iDy  Abel  and  Ford  to  be  a  glucoside,  and  thus  belongs  to  the  saponin 
group.  (See  Chap.  vi.  for  further  discussion.)  In  the  leaves  of  the  ivy,  Hedera 
helix,  a  hemolytic  glucoside  has  been  found  by  Moore. i-  It  is  of  interest  that 
Faust  believes  the  poisonous  agent  of  cobra  venom  to  be  a  glucoside,  closely  re- 
sembling sai^otoxin. 

As  will  be  seen,  all  these  last-mentioned  vegetable  hemolytic  agents 
are  essentially  different  from  either  the  bacterial  or  scrum  hemol.ysins, 
or  from  the  abrin,  ricin,  crotin,  or  robin  group,  in  that  the}'  are  of 
relatively  simple  chemical  composition,  and  quite  unlike  proteins,  en- 
zymes, or  toxins.  The  manner  in  which  they  cause  hemol^'sis  is 
unknown,  but  from  their  relation  to  saponin  it  is  probable  that,  like 

'•>  Jour.  Path,  and  Bact.,  1910  (15),  56. 
6  Arch.  exp.  Path.  u.  Pharm.,  1SS7  (23),  233. 

'Literature,  see  Meyer  and  Schmiedeberg,  Arcii.  f.  exi).  Path.  u.  Pharm.,  ISOo 
(36),  361;  Perles,  ibid.,'\mO  (2{)),    SS. 

*  See  Kunkel,  "Handbuch  der  Toxokologie,"  p.  873. 

"  Concerning  human  solanin  jjoisoning  see  liothe,  Zeit.  f.  Hyg.,  l*.)19  (88),  1. 
lOBoehm  and  Kiilz,  Arch.  exp.  Path.  u.  Pliarm.,  1S85  (19),  -103. 

11  Deut.  Arch,  kliii.  Med.,  1883  (32),  209. 

12  Jour.  Pharnuicol.,  1913  (4),  263.   . 


HEMOLYSIS  BY   VENOMS  223 

it,  they  cause  injury  by  coniljinin^  witli  or  dissolvinji;  the  lijjoids  of 
the  stroma  of  the  corpuscles.  Extracts  of  Morchella  esculenta  do  not 
hemolyze  corpuscles  in  vitro,  although  powerfully  heiiioh'tic  when 
injected  into  animals,  and  causing  severe  hemoglobinuria;  so  that 
it  is  probable  that  they  cause  their  hemolj'tic  effects  indirectly  through 
the  changes  which  they  produce  in  the  tissues  of  the  poisoned  animal.'-^ 

Hemolysis  by  Venoms'^ 

The  laking  of  blood-corpuscles  by  venoms  is  of  peculiar  interest 
from  the  standpoint  of  immunity  phenomena,  since  it  was  demon- 
strated by  Flexner  and  Noguchi  that  the  hemolytic  principle  of  the 
venoms  resembles  an  amboceptor,  in  that  some  substance  behaving 
like  complement  has  to  be  furnished  by  the  blood.  Kyes  found 
that  this  complementing  agent  is  lecithin,^"  and  was  able  to  produce 
what  he  considers  to  be  compounds  of  the  hemolysin  with  lecithin, 
called  "lecithids."  The  hemolytic  activity  of  these  lecithids  is  very 
great,  and  they  seem  to  be  free  from  the  neurotoxic  principle  of  the 
venoms.  Whether  they  represent  true  compounds  of  a  hemoh'tic 
amboceptor  with  lecithin,  or  are  simply  actively  hemolytic  products 
of  the  cleavage  of  lecithin  by  an  enzymatic  activity  of  the  venom,  is 
at  present  unsettled ;^^  it  seems  probable,  however,  that  the  hemolysin 
of  cobra  venom  is  a  lipase  that  splits  lecithin  into  two  hemoh'tic 
components,  oleic  acid  and  " desoleolecithin "  (Coca).'^  Noguchi 
suggests  that  not  only  lecithin,  but  also  soaps,  especially  of  unsaturated 
fatty  acids,  and  probably  protein  compounds  of  soaps  and  lecithin, 
may  act  as  the  hemolytic  "complement"  which  activates  venoms. 
The  hemolytic  agents  of  venom  seem  to  be  secreted  by  the  salivarj- 
glands  of  the  reptiles  from  their  blood,  which  contains  almost  identical 
amboceptors,  differing  chiefly  in  that  they  can  be  activated  onl}'  by 
agents  contained  in  snake  blood,  while  the  amboceptors  of  venom  can 
be  activated  b}-  nearlj'  all  sorts  of  blood.  Venoms  from  cobra,  rattle- 
snake, moccasin,  and  copperhead  possess  in  each  a  varietj'  of  inter- 
mediary bodies  (amboceptors)  that  seem  to  be  at  least  partly  identical 
in  nature,  although  they  may  varj^  in  quantity.  In  order  of  decreasing 
hemolytic  power  for  mammalian  corpuscles  come  venoms  from  cobra, 
water  moccasin,  copperhead,  and  rattlesnake.  These  venoms  are 
also  agglutinative  for  all  corpuscles  tried,  and  agglutination  will 
occur  at  0°  C.  Exposure  for  thirty  minutes  at  75°-80°  C.  destroys 
the  agglutinating  property.     In  general,  the  hemolytic  power  of  the 

1'  Friedberger  and  Brossa,  Zeit.  Immunitat.,  1912  (15),  506. 

'^  General  review  of  literature  on  the  hemolytic  properties  of  animal  poisons 
given  bv  Sachs,  Biochem.  Centralblatt.  1906  (5),  257;  Noguchi,  Jour.  Exp.  IMed., 
1907  (9),  436. 

15  Cruickshank  also  found  that  other  lipoids  than  lecithin  may  activate  cobra 
venom  (Jour.  Path,  and  Bact.,  1913  (17),  619). 

16  See  Kyes,  Jour.  Infect.  Dis.,  1910  (7),  181;  v.  Dungern  and  Coca,  ibid.,  1912 
(10),  57;  Manwaring,  Zeit.  Immunitat,  1910  (6),  513;  Bang,  iUd.,  1910  (8),  202; 
Coca,  Jour.  Infect.  Dis.,  1915  (17),  351. 


224  CHEMISTRY'  OF  THE  IMMUNITY  REACTIONS 

venoms  for  different  sorts  of  corpuscles  varies  in  inverse  propor- 
tion to  their  agglutinative  power.  The  hemolytic  intermediary  bodies 
are  resistant  to  heat,  suffering  but  slight  loss  of  power  at  100°  C. 
Red  corpuscles  of  the  frog  are  not  hemolyzed  by  venom,  and  those  of 
necturus  (mud  pupp}')  but  slightlj',  agreeing  with  the  known  resist- 
ance of  cold-blooded  animals  to  snake-bites. 

The  erythrocj^tes  of  different  individuals  show  considerable  varia- 
tions in  their  resistance  to  hemolytic  agents,  perhaps  depending  upon 
tile  amount  or  upon  the  manner  of  fixation  of  the  lipoids  in  the  cor- 
puscles; thus  the  corpuscles  of  syphilitics  show  a  heightened  resist- 
ance to  hemolysis  by  cobra  venom  (Weil)'^  except  in  the  earliest 
stages,  when  they  are  hypersensitive.  Also,  the  serum  of  persons  suf- 
fering from  various  diseases,  especially  mental  diseases,  inhibits  the 
hemolysis  of  human  corpuscles  by  cobra  venom. '^  After  splenectomy 
there  is  an  increased  resistance  to  venom  hemolysis.'^ 

Eel  serum  is  remarkably  hemolytic,  so  much  so  that  a  quantity'  of  0. 1  c.c.  per 
kilogram  of  body  weight  will  kill  a  rabbit  or  guinea-pig  in  three  minutes  when 
injected  intravenously.  Heating  at  54°  C.  for  fifteen  minutes  destroys  the  hemo- 
lytic action,  and,  unlike  ordinary  serum  hemolysins  the  addition  of  complement 
does  not  restore  its  activity.  Animals  can  be  immunized  against  this  serum.  In- 
troduced into  the  stomach  in  ordinary  quantities  eel  serum  is  not  to.\ic.  It  can 
be  dried  and  redissolved  without  losing  its  activity,  but  acids  and  alkalies  readilj' 
destroy  it.  Mosso,  who  first  discovered  the  toxicity  of  eel  serum,  called  the  un 
known  active  principle  ichthyotoxin.  It  is  found  chiefly  in  the  albumin  fraction 
of  the  eel  serum. ^^  Many  other  animals  produce  hemolytic  poisons  (e.  g.,  spiders, 
bees)  which  are  discussed  under  Zootoxins,  Chapter  vi. 

Hemolysis  in  Disease 

During  health  there  is  alwaj^s  going  on  a  certain  amount  of  de- 
struction of  red  corpuscles  that  have  outlived  their  usefulness;  hence 
in  disease  we  may  have  to  deal  with  either  an  alteration  in  the  nor- 
mal processes  of  blood  destruction  or  the  introduction  of  entirely  new 
processes.  Although  the  place  and  manner  of  normal  red  corpuscle 
destruction  is'inot  completel}^  known,  yet  it  seems  probable  that  there 
is  relatively  little  hemolysis  within  the  circulating  blood.  "When  a 
red  corpuscle  becomes  damaged,  it  seems  to  become  more  susceptible 
to  phagocytosis,  and  it  is  then  picked  out  of  the  blood,  chiefl}'  by  the 
endothelial  cells  of  the  sinuses  of  the  liver,  spleen,  hemolymph  glands, 
and  bone-marrow.  Within  these  cells  it  apparently  undergoes  hemo- 
lysis. Eventually,  the  resulting  pigment  is  split  up  b}'  the  liver,  the 
non-ferruginous  portion  forming  the  bile-pigments,  while  the  iron 
seems  to  be  mostly  withheld  to  be  worked  over  into  new  hemoglobin.-' 

>'  Jour.  Infect.  Dis.,  1909  (6),  688;  Stone  and  Schottstaedt,  Arch.  Int.  Med., 
1912  (10),  8. 

*"  See  articles  on  this  subject  in  the  Miinch.  med.  Woch.,  1909,  Vol.  56. 

1"  Kolmer,  Jour.  Exp.  Med.,  1917  (25),  195. 

2»Sato,  Kyoto  .Jour.  Med.  Sci.,  1917  (14),  36. 

21  Muir  and  Dunn  (.lour.  Path,  and  Bact.,  1915  (20),  41),  find  that  after 
acute  hemolytic  anemia  in  ral)bits  the  excess  iron  stored  in  the  organs  lias  been 
nearly  all  aljsorbed  by  the  time  regeiuMatioii  of  tlu>  blood  is  complete. 


HEMOLYSIS   l.\   DISKASE  225 

(Seo  "Pisincntutioii,"  (hap.  xviii.)  Whoiicvcr  (luring  disca^;  red 
corpuscles  arc  more  rapidly  injured  than  they  are  under  normal  condi- 
tions, these  processes  of  normal  hemolysis  are  exaggerated  and  we  not 
only  find  the  phagocytic  cells  of  the  spleen  and  glands  packed  with 
corpuscles,  but  endothelial  cells  elsewhere,  and  also  leucocytes,  take 
on  the  hemolytic  function.  At  the  same  tinie  there  results  an  exces- 
sive production  of  bile-pigment  from  the  destroyed  red  corpuscles, 
which  has  an  etiological  relation  to  the  so-called  "hemato-hepatogen- 
ous"  jaundice.  If  hemolysis  is  very  excessive,  the  blood  pigment 
accumulates  in  other  organs  than  the  liver  and  spleen.  According  to 
Pearcc^^  and  his  associates,  when  the  blood  contains  at  one  time  more 
than  0.06  gm.  of  free  hemoglobin  per  kilo  of  body  weight,  it  begins  to 
be  excreted  by  the  kidneys;  smaller  amounts  are  cared  for  chiefly 
by  the  liver,  and  even  when  much  larger  amounts  of  hemoglobin  are 
present  in  the  blood  the  liver  takes  care  of  most  of  it,  only  a  rela- 
tively small  proportion,  17  to  36  per  cent,  being  excreted  in  the  urine. 
Hence  it  is  possible  to  have  hemolytic  jaundice  without  hemoglobin- 
uria. Part  of  the  pigment  is  converted  into  urobilin,  and  the  amount 
of  this  pigment  in  the  stool  is  an  index  of  the  amount  of  hemolysis.-^ 
In  persons  with  hemolytic  hemoglobinemia,  intravenous  injection  of 
hemoglobin  will  produce  hemoglobinuria  with  smaller  dosage  than  in 
normal  persons,  who  require  at  least  17  c.c.  of  laked  corpuscles  to  pro- 
duce hemoglobinuria.-^ 

It  is  possible  that  the  globin,  which  is  quite  toxic  when  free,'^  may 
play  a  part  in  the  symptomatology  of  hemolytic  poisons.  The  stroma 
of  the  erythrocytes  also  seems  to  be  toxic. ^^ 

The  hemolj^sis  of  the  acute  febrile  diseases  is  readily  explained  by  the 
demonstrable  hemolytic  property  of  the  products  of  the  organisms 
that  cause  them,  such  as  streptocolysin,  staphylolysin,  etc.  Perhaps 
at  the  same  time  products  of  altered  metabolism  may  also  play  a 
part,  but  it  does  not  seem  probable  from  experimental  results  that 
the  thermic  condition  per  se  has  much  effect.  In  malaria,  although 
the  parasites  enter  and  destroy  the  corpuscles  in  which  they  live,  yet 
this  alone  does  not  account  for  all  the  blood  destruction  of  the  disease, 
for  the  amount  of  anemia  is  quite  without  relation  to  the  number 
of  parasites  to  be  found.  There  is  good  reason  to  believe  that  the 
Plasmodia  produce  hemolytic  substances  that  are  chschargcd  into  the 
serum. 

In  the  primary  anemias  hemolj^sis  seems  to  be  the  essential  process, 
although  the  agents  involved  are  at  present  unknown.  Absorption 
of  hemolytic  products  of  intestinal  putrefaction  or  infection  has 
always  come  in  for  much  suspicion,  without  ever  becoming  completely 

"Jour.  Exp.  Med.,  1912  (16),  several  articles. 

"  See  Robertson,  Arch.  Int.  Med.,  1915  (15),  1072. 

"  Sellards  and  Minot,  .Jour.  Med.  Res.,  1916  (34),  469. 

"  Schittenhelin  and  Weichardt,  Miinch.  med.  Woch.,  1912  (59),  1089. 

-«  Barratt  and  Yorke,  Brit.  Med.  Jour.,  Jan.  31,  1914. 


226  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

established.  Here  also  the  hemolysis  seems  to  take  place  in  the  endo- 
thelial cells  rather  than  in  the  vessels.  In  such  a  disease  as  pernicious 
anemia  there  is  much  reason  to  assume  that  defective  or  abnormal 
hematogenesis  is  an  important  factor.  Probably  the  anemia  of 
nephritis  is  at  least  partly  the  result  of  hemolytic  action  of  the 
retained  products  of  metabolism,  in  which  connection  the  hemolytic 
properties  of  ammonium  compounds  may  be  recalled.  In  some  diseases 
associated  with  anemia  it  has  been  found  that  the  blood-serum  of  the 
patient  is  cUstinctly  isohemolytic,  although  isoagglutination  seems  to 
be  more  frequent.  The  fluids  that  can  be  obtained  from  cancers  have 
been  found  to  be  hemolytic,  while  antihemolysin  has  been  found  in 
ascitic  and  pleural  effusions.  Autolytic  disintegration  of  hver,  and 
presumably  other  tissues,  may  also  cause  the  presence  of  hemolytic 
substances  in  the  blood. ^^  Arseniuretted  hydrogen  may  produce 
hemolysis  in  some  such  way,  since  it  causes  no  hemolj^sis  in  the  test 
tube  (Heffter).  The  very  great  hemolytic  action  of  soaps  and  free 
fatty  acids,  which  varies  directly  with  the  number  of  unsaturated 
carbon  atoms  they  contain  (Moore^^),  makes  it  possible  that  these 
substances  play  a  part  in  the  hemolysis  of  disease,  especially  since  the 
fatty  acids  of  the  liver  are  characterized  by  their  high  content  of  free 
fatty  acids.  Bile  is  strongly  hemolytic,  and  in  icterus  this  is  an  im- 
portant consideration. 

In  many  forms  of  poisoning  hemolysis  is  a  prominent  feature;  in 
some  it  seems  to  be  the  chief  effect  of  the  poison,  e.  g.,  potassium 
chlorate  and  arseniuretted  hydrogen.  In  severe  extensive  burns  there 
may  occur  hemolysis,  and  hemoglobinuria  may  also  result.  The  hemo- 
globinuria of  "blackwater  fever"  has  been  the  cause  of  much  discus- 
sion as  to  whether  the  malarial  parasite  or  the  quinine  is  the  cause, 
with  a  divided  opinion  resulting,  although,  undoubtedly,  cases  do  occur 
in  malaria  without  administration  of  quinine.  The  studies  of  Brem-® 
indicate  that  the  hemolysis  is  produced  by  a  hemolysin  coming  from 
the  Plasmodium,  and  that  the  quinine  influences  the  condition  by 
preventing  the  action  of  an  antihemolytic  substance  present  in  the 
blood. 

After  removal  of  the  spleen,  hemolysis  by  the  hemolymph  glands 
exceeds  that  of  the  primitive  spleen,  causing  an  excessive  destruction 
of  red  corpuscles  (Warthin^").  This  suggests  that  the  spleen  may 
normally  dispose  of  some  hemolytic  agent  which  acts  either  by  stimu- 

"  Maidoni,  Biochoin.  Zeit.  ,1912  (45),  328.  lieinolytic  lipoids  are  believed  by 
some  to  l)e  liheruted  from  injured  tissues  (see  Kirsche,  Hiochem.  Zeit.,  1913  (55), 
169),  but  McPhedran  (Jour.  Exp.  Med.,  1913  (18),  527)  could  find  no  evidence 
that  any  particularly  hemolytic  fatty  ucid,  more  active  than  oleic  acid,  can  be 
isolated  from  either  normal  or  diseased  tissues. 

"^^  Brit.  Med.  Jour.,  1909  (ii),  684;  sec  al.so  Lamar,  Jour.  Exper.  Med.,  1911 
(13),  380. 

2»  Arch.  Int.  Med.,  1912  (9),  129. 

a"  Jour.  Med.  Research,  1902  (7),  435. 


PAROXYSMAL  HEMOGLOBINURIA  227 

lating  phagocytosis  or  by  so  altering  the  red  cells  that  they  are 
particularly  susceptible  to  phagocytosis.  This  idea  is  not  substanti- 
ated by  the  work  of  Pearce,^^  who  found  the  anemia  of  splenectomy 
accompanied  by  an  increased  resistance  of  the  corpuscles  to  hemolysis, 
and  no  hemolytic  agent  was  present  in  the  blood.  There  also  occurs 
the  group  of  anemias  associated  with  great  enlargement  of  the  spleen, 
and  in  which  removal  of  the  spleen  may  result  in  a  return  to  nofmal 
blood  conditions;  a  fact  suggesting,  among  other  possibilities,  that 
there  may  be  poisons  which  stimulate  directly  the  hemolytic  action 
of  the  spleen  independent  of  the  natural  stimulation  of  splenic  hemoly- 
sis which  comes  from  the  presence  in  the  splenic  blood  of  injured  red 
corpuscles. ^- 

Resistance  to  hemolysis  varies  greatly  in  disease  conditions'^  and  often  specific- 
ally,— i.e.,  resistance  may  be  increased  to  one  agent,  decreased  for  another,  and 
normal  with  a  third.  Attempts  have  been  made  to  use  this  resistance  as  a  diag- 
nostic or  prognostic  index,  but  not  with  great  success  in  most  cases.  Apparently 
changes  in  the  plasma  lead  to  alterations  in  the  permeability  of  the  corpuscles, 
which  determines  their  behavior  with  hemolytic  agents;  also  changes  in  the  pro- 
portion of  lipoids  and  hemoglobin  may  modify  hemolysis.  As  an  example  of 
this  condition  may  be  cited  observations  on  hemolysis  by  cobra  venom,  the  cor- 
puscles having  been  found  less  resistant  in  dementia  precox,  more  resistant  in 
carcinoma  and  syphilis.  Butler"  states  that  fragility  of  the  corpuscles  is  abnor- 
mally high  in  exophthalmic  goiter,  cancer,  syphilis,  tabes,  anemia  and  malaria. 
In  obstructive  jaundice  the  corpuscles  show  an  increased  resistance  to  hemolysis 
by  hypotonic  salt  solution,  but  in  congenital  hemolytic  jaundice  the  resistance  is 
decreased.'^  Using  saponin  hemolysis,  Bigland'^  found  the  resistance  greatly 
decreased  in  icterus,  although  the  serum  had  an  increased  protective  action  because 
of  antagonism  between  the  saponin  and  the  bile  salts;  in  all  anemias  resistance 
was  found  increased,  except  pernicious  anemia,  which  showed  normal  or  slightly 
subnormal  resistance;  high  temperature  decreases  resistance.  As  will  be  seen  from 
the  few  examples  cited,  the  resistance  to  different  hemolj'tic  agents  may  vary  with 
the  same  corpuscles.'^ 

Paroxysmal  Hemoglobiniiria.^^ — This  condition  seems  to  depend 
upon  the  presence  in  the  serum  of  a  hemolytic  amboceptor  (an  auto- 
hemolysin) ,  which  will  combine  with  the  corpuscles  of  the  same  indi- 
vidual and  sensitize  them  for  his  own  complement  (Donath  and 
Landsteiner,  Eason) .  This  au^  ohemolysin  can  react  with  the  corpuscles 
only  at  low  temperature,  such  as  may  be  furnished  in  the  peripheral 
vessels  by  exposure  to  cold,  and  the  complement  unites  when  the  tem- 
perature of  these  corpuscles  again  reaches  37°  in  other  parts  of  the 

'^  Pearce  ct  al,  Jour.  Exp.  Med.,  1912,  ^'ol.  16.  See  also  Roccavilla,  Arch. 
Med.  Exp.,  1915  (26).  508. 

"  See  Banti.  Semain  Med.,  1913  (33),  313. 

'3  Keview  bv  Paltauf,  Krehl  and  Marchand's  Handb.  allg.  Pathol.,  1912  (II 
(1),)  83. 

'*  Quarterly  Jour.  Med.,  1913  (6),  145. 

"  See  Richards  and  Johnson,  Jour.  Amer.  Med.  Assoc,  1913  (51),  1586  Giffin 
and  Sanford.  Jour.  Lab.  Clin.  Med.,  1919  (4),  465. 

'6  Quart.  Jour.  Med.,  1914  (7),  370. 

"  Bibliography  by  Krasny,  Folia  Hematol.,  1913  (16),  353. 

''  Landsteiner,  Handbuch  d.  Biochem.,  Vol.  2  (1),  p.  492;  Meyer  and  Emmerich, 
Deut.  Arch.  klin.  Med.,  1909  (96),  287. 


228  CHEMISTRY  OF  THE  IMMVXITY  REACTIOXS 

body.  In  susceptible  persons  attacks  of  hemoglobinuria  may  be 
brought  on  merely  by  holding  the  hands  in  cold  water,  and  their  blood 
serum  will  sensitize  to  hemolysis  human  corpuscles  (even  of  normal 
individuals),^^  m  vitro  at  low  temperatures.""*  Certain  infections,  es- 
pecially syphilis, ^^  predispose  to  paroxysmal  hemoglobinuria.  Not 
only  the  hemolytic  amboceptors,  but  also  an  auto-opsonin  is  present 
(Eason)  and  the  resistance  of  the  red  corpuscles  is  decreased  to  various 
harmful  agencies,  including  COo  and  other  weak  acids.*-  The  cor- 
puscles of  three  cases  studied  by  Moss'*^  showed  an  increased  resistance 
to  hypotonic  NaCl  solutions.  Just  before  the  rigor,  hemolysins  may 
be  found  in  the  blood,  disappearing  after  the  hemoglobinuria  (Rob- 
erts).** In  a  case  studied  by  Dennie  and  Robertson,*^  hematuria 
resulted  from  destruction  of  only  6.3  c.c.  of  the  patient's  blood,  and 
90  per  cent,  of  the  liberated  hemoglobin  was  excreted  within  two  hours. 
There  also  occur  conditions  in  which  aiito-aggluti7iati on  occurs  without 
hemolysis  when  the  blood  is  cooled.*'' 


Pathological  Anatomy  in  Hemolysis. — The  lesions  produced  in  the  organs  of 
animals  poisoned  with  hemolytic  agents  are  usually  pronounced  and  quite  char- 
acteristic. There  is  often  a  subcutaneous  edema,  which  is  usually-  blood-stained, 
and  similar  fluid  may  be  present  in  the  serous  cavities.  The  fat  is  yellowish,  and 
the  muscles  are  darker  in  color  than  is  normal.  The  spleen  is  usually  much 
swollen,  soft,  friable,  and  very  dark  in  color.  The  liver  is  usually  swollen  and 
mottled  with  red  areas  in  a  yellow  background.  The  renal  cortex  is  dark  in 
color,  even  chocolate-colored,  and  the  pyramids  are  comparatively  light;  hemo- 
globin is  frequentlj'^  present  in  the  urine.  In  the  lungs  are  often  found  hemor- 
rhages or  areas  resembling  small  infarcts.  The  blood  may  be  thin  and  even 
distinctly  transparent.  Microscopically  the  red  corpuscles  are  found  in  all  condi- 
tions of  degeneration,  and  often  fused  together.  In  the  liver,  besides  patches  of 
congestion,  fatty  changes  are  present  if  the  animal  lives  long  enough.  Large 
phagocytic  cells  packed  with  red  corpuscles  are  abundant  in  the  spleen  and  lymph- 
glands,  as  well  as  diffuse  accumulations  of  the  blood-cells,  which  are  often  fused; 
and  much  pigment  is  also  present,  both  free  and  in  the  cells.  Pigment  also  accum- 
ulates in  the  renal  epithelium,  which  often  shows  much  disintegration;  congestion 
is  prominent,  and  hemorrhages  into  both  interstitial  tissue  and  glomerules  are 
frequent.  Some  of  the  lesions  are  due  to  the  hemolysis,  and  some  to  the  associated 
agglutination  of  corpuscles,  which  form  hyaline  thrombi.  Pearce""  lias  found  that 
agglutinative  serum  when  injected  into  dogs  causes  widespread  necrosis  in  the 
liver,  which  is  followed  by  proliferation  of  connective  tissue  and  the  production 
of  changes  re.seml)ling  cirrhosis.  There  is  a  marked  decrease  in  the  glycogen 
content  of  the  liver,  and  of  its  lipolytic  activity'  (Andrea).'" 

39  See  Lorant,  Deut.  Arch.  klin.  Med.,  1918  (126),  148. 

*"  Widal  looks  upon  paro.\ysmal  hemoglobinuria  as  an  autoanajjliviaxis  (Semain 
M6d.,  1913  (33),  013). 

*'  Matsuo,  Arch.  f.  klin.  Med.,  1912  (107)  335. 

«  lierghausen,  Arch.  Int.  Med.,  1912  (9),  137. 

"  Folia  Serologica,  1911  (7),  1117. 

**  Brit.  Med.  Jour.,  1915  (2),  398. 

*'  Arch.  Int.  Med.,  1915  (lb),  205. 

'"' See  Rous  and  Robertson,  Jour.  Exp.  Med.,  191N  (27^,  .")()3;  C'lough  and 
Richter,  Hull.  Johns  Hop.  Hosp.,  191S  (29),  80. 

"".Jour.  Kx]).  Med.,  1900  (8),  04;  Jour.  Med.  IJesearcli.   1900  (14).  541. 

*'  Arch,  internal.  i)harmacodyn.,  1905  (14),  177. 


COMPLEMENT  FIXAT/(>.\   liKM'TlOS  229 

COMPLEMENT  FIXATION"'  AND  WASSERMANN  REACTIONS'' 

The  original  principle  involved  in  these  reactions  was  first  demon- 
strated by  Bordet  and  Gengoii,  and  is  essentially  as  follows:  If  a 
specific  antigen  and  amboceptor  unite  in  the  presence  of  complement, 
the  complement  is  then  united  to  the  amboceptor-antigen  compound 
to  complete  the  reaction.  When  sufficient  amounts  of  amboceptor 
and  antigen  are  present  the  entire  quantity  of  available  complement 
may  be  thus  fixed,  and,  consequently,  the  mixture  contains  no  more 
complement  for  further  reactions.  As  complement  does  not  ordinarily 
unite  with  amboceptors  except  when  the  amboceptors  are  united  with 
their  specific  antigens  the  fact  that  in  a  given  system  of 

complement  +  amboceptor  +  antigen 
\ 
there  is  no  free  complement,  is  evidence  of  a  reaction  between  ambocep- 
tor and  antigen;  in  consequence  of  which  this  reaction  can  be  used  to 
determine  the  presence  of  a  specific  amboceptor  in  a  serum^  by  using 
the  corresponding  antigen;  or  conversely,  with  a  scrum  containing  a 
known  amboceptor  we  can  detect  the  presence  in  a  solution  of  the 
specific  antigen.  The  indicator  of  the  presence  or  absence  of  comple- 
ment which  is  in  universal  use,  is  the  ability  of  the  mixture  to  hemolyze 
erythrocytes  in  the  presence  of  the  specific  hemolytic  amboceptor. 
Thus,  if  typhoid  bacilli  and  a  typhoid  antiserum  which  contains  both 
complement  and  specific  amboceptor,  are  mixed  in  proper  proportions 
and  incubated  for  a  short  time,  the  complement  will  be  bound  to  the 
bacilli.  If  we  then  add  this  mixture  to  sheep  corpuscles  which  have 
been  acted  upon  by  an  antisheep-corpuscle  serum,  from  which  the 
complement  had  been  previously  removed  by  heating,  no  hemoh'sis 
will  occur,  for  we  have  added  no  free  complement.  But  if  our  original 
mixture  had  contained  dysentery  bacilli  instead  of  typhoid  bacilli 
the  complement  would  not  have  been  fixed,  and  the  addition  of  this 
mixture,  containing  free  complement,  to  the  sensitized  sheep  corpuscles 
would  cause  prompt  hemolysis. 

This  reaction  was  at  first  used  for  the  detection  of  antibodies  in 

■•^  The  reaction  of  ''complement  fixation"  must  not  be  confused  with  the  eu- 
tirelj'  distinct  reaction  of  ''complement  deviation,"  a  mistake  very  likely  to  ha])pen 
because  of  the  careless  but  erroneous  use  by  some  writers  of  the  latter  term  in 
describing  the  first-named  reaction.  Complement  deviation  (or  Neisser-Wechs- 
berg  phenomenon)  is  produced  when  an  excess  of  amboceptors  is  present  together 
with  antigen  and  a  limited  amount  of  complement,  which  results  in  absence  of 
complement  activitv.  The  mechanism  of  this  reaction  lias  not  been  satisfactorily 
explained.  Thjotta  (Norsk.  Mag.  Laegevid.,  1919  (80),  1051)  believes  it  to  de- 
pend on  some  special  substance,  distinct  from  the  known  antibodies,  which  adsorbs 
complement. 

■"Literature  given  by  Meier,  Jahresbor.  d.  Immunitatsforsch.,  1909  (4),  58; 
Sachs  and  Altmann,  Kolle  and  Wassermann's  Handbuch,  Ergiinzungsbd..  2,  1909, 
p.  476;  Noguchi,  "Serum  Diagnosis  of  Svphilis  and  Luetin  Reaction,""  I'hiladel- 
phia,  1912. 


230  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

sera,^°  and  for  the  identification  of  bacteria,  and  was  found  to  be  ex- 
quisitely delicate,  detecting  most  minute  amounts  of  antigens  with 
the  sharpest  specificity  limits  of  any  of  the  immunity  reactions.  On 
account  of  the  delicacy  of  this  reaction  it  can  be  used  to  determine 
the  presence  in  tissues  of  specific  organisms  which  cannot  be  culti- 
vated; thus,  it  has  been  possible  to  demonstrate  the  existence  of  a 
specific  scarlatinal  virus^^  in  the  tissues  during  this  disease,  although 
the  actual  organism  cannot  be  isolated.  This  fact  led  Wassermann 
to  use  extracts  of  the  livers  of  congenital  sj'-philitic  fetuses,  which 
contain  great  quantities  of  spirochetes,  as  an  antigen  for  complement 
fixation  reactions,  whereby  it  should  be  possible  to  determine  in  a 
given  serum  the  presence  of  specific  amboceptors  for  the  virus  of 
syphilis,  such  amboceptors  being  present  in  persons  infected  with 
syphilis  as  a  result  of  the  reaction  to  the  infection.  As  originally  in- 
troduced, then,  the  Wassermann  reaction  was  supposed  to  be  simply 
a  specific  reaction  between  syphilitic  antigen,  specific  syphilitic  am- 
boceptors, and  non-specific  complement.  It  was  soon  learned,  how- 
ever, that  the  reaction  as  it  occurred  in  syphihs  was  decidedly 
different  from  the  original  complement  fixation  reaction  of  Bordet 
and  Gengou,  for  it  was  found  possible  to  substitute  in  the  reaction  for 
extracts  of  tissues  containing  syphilitic  virus  (spirochetes),  the  most 
varied  sorts  of  tissue  extracts,  coming  from  tissues  certainly  free 
from  spirochetes  (e.  g.,  ox  heart).  Noguchi  and  Bronfenbrcnncr^^ 
summarize  the  present  state  of  the  matter  in  these  words:  "We  know 
merely  this:  that  complement  in  the  presence  of  syphilitic  antigen 
may  be  rendered  inactive  by  one  or  more  substances  in  the  body 
fluids  of  a  syphilitic  or  parasyphilitic  patient." 

Extended  investigation  of  these  non-specific  antigens  which  give 
specific  complement  fixation  with  S3^philitic  sera,  has  shown  them  to 
be  related  to  the  lipoids,  especially  the  lecithins,  as  indicated  by  the 
fact  that  the  most  efficient  antigens  contain  the  accton-insolublo  frac- 
tion of  the  tissue  lipoids.  The  antigenic  value  of  this  fraction  of 
different  liver  extracts  varies  nearly  directly  with  its  power  to  com- 
bine with  iodin*^  (Noguchi  and  Bronfenbrenncr),  which  indicates 
that   the   unsaturated  fatty   acids   arc   important  in   the   reaction.^'' 

'S"  Accoi-fling  to  Gay  (Univ.  of  Calif.  Publ.,  Piithol.,  1911  (2),  1,  full  discus- 
sion) conipleinent  fixation  i.s  produced  by  an  antincn-antihody  I'oinjjlex  distinct 
from  precipitinof^en-precipitin,  but  Dean  (Zeit.  f.  Ininumitat.,  1912  (13),  81)  be- 
lieves that  they  represent  two  phases  or  stages  of  the  same  reaction.  Thiele  and 
Embleton  (Zeit.  luuaunitat.,  1913  (10),  430)  consider  tliat  in  syphilis  it  is  not 
a  specific  antibody,  but  an  anti-complementary  substance  which  arises  from  the 
disintegrating  tissiies. 

^'  Koesslerand  Koessler,  Jour.  Infec.  Dis.,  1912  (9),  3GG. 

"  Jour.  Exp.  Med.,  1911  (13),  43. 

'^  Not  corroborated  by  Hrowning,  Cruickshank  and  (iilmour.'''' 

^*  An  interesting  observation  made  l)y  Noguchi  and  Hrt)nfenbrenner,  is  that  ex- 
tracts from  fatty  livers  are  alnut.st  devoid  of  antigenic  projuMties;  but  So  (Cent.  f. 
Bakt.,  1912  (03).  43.S)  found  that  the  extract  from  fatty  hearts  of  guinea-pigs 
was  more  active  than  from  normal  hearts. 


COMPLEMENT  FIXATION  REACTION  231 

Cnule  lecithins  from  different  sources  vary  in  efficiency,  heart  lecithin 
being  more  active  than  liver  lecithin,  brain  and  egg  yolk  lecithin  follow- 
ing. Pure  lecithin  is  not  effective,  the  activity  of  lipoid  solutions 
depending  upon  some  other  substance  which  is  difficult  to  separate 
from  lecithin  (MacLean).^^  Addition  of  cholesterol  to  the  lipoid 
solutions  increases  greatly  their  activity. ^^  An  acetone-precipitated 
"antigen"  of  this  class  is  not  a  true  antigen,  however,  for  fixation 
antibodies  are  not  developed  in  animals  injected  with  such  a  lipoid 
which  has  been  shown  to  be  entirely  efficient  in  the  Wassermann 
reaction.  ^^ 

As  for  the  substance  in  the  syphilitic  serum  which  participates  in 
the  Wassermann  reaction,  it  would  seem  to  be  related  to  the  globulins, 
which  are  decidedly  increased  in  the  blood  and  spinal  fluid^''  of  syphili- 
tics,^**  especially  the  euglobulin.^''  P.  Schmidt^'  ascribes  the  reaction 
to  the  physico-chemical  properties  of  the  globulins  of  the  syphilitic 
serum,  which,  he  believes,  possess  a  greater  affinity  for  the  colloids 
of  the  antigen  than  normal  globulins;  this  affinity  is  held  in  check  in 
normal  serum  by  the  albumins  of  the  serum,  which  are  relatively  or 
absolutely  decreased.  That  physico-chemical  factors  do  play  a  part 
is  evidenced  by  the  common  observation  that  the  turbidity  of  the 
antigen  suspension  is  closely  related  to  its  efficiency,  clear  solutions 
being  less  active.  Slight  changes  in  H-ion  concentration  will  change 
a  reaction  from  negative  to  positive,  or  reverse;  and  neutral  salts  can 
change  a  negative  to  a  positive  reaction,  but  not  the  reverse  (Gumm- 
ing.) "^^  The  lipoids  in  syphilitic  sera  are  said  by  Peritz^^  to  be 
increased,  but  the  lipoid  content  and  the  antibody  titer  do  not  show 
any  constant  relation  (Bauer  and  Skutezky).''^  The  cholesterol  con- 
tent of  syphilitic  blood  shows  no  evidence  of  a  quantitative  relation  tb 
the  Wassermann  reaction. ^^  Friedemann^^  believes  that  a  globulin- 
soap  compound  is  the  active  substance  in  syphilitic  sera.  Mcintosh" 
says  that  the  active  component  differs  from  typical  antibodies  in  not 

^^  Monographs  on  Biochemistry,  "Lecithin  and  Allied  Substances,"  London, 
1918. 

5"  Browning  et  al,  Zeit.  Immunitat.,  1912  (14),  284;  Jour.  Pathol,  and  Bact., 
1911  (16),  135  and  225.  Klein  and  Fraenkel  believe  the  "antigen"  of  ox  heart 
extracts  to  be  a  combination  of  lecithin  with  cholesterol  and  small  amounts  of 
a  soap-like  substance  similar  to  jecorin  (Miinch.  med.  Woch.,  1914  (61),  651.) 

"  Fitzgerald  and  Leathes,  Univ.  of  Calif.  Publ.,  Path.,  1912  (2),  39. 

«8  Pfeiffer,  Kober  and  Field,  Proc.  Soc.  Exp.  Biol.,  1915  (12),  153. 

"  See  Rowe,  Arch.  Int.  Med.,  1916  (18),  455. 

««  MuUer  and  Hough,  Wien.,  klin.  Woch.,  1911  (24),  167. 

"  Zeit.  f.  Hyg.,  1911  (69),  513.  See  also  Hirschfeld  and  Klinger,  Zeit.  Immuni- 
tat., 1914  (21),  40. 

"  Jour.  Infect.  Dis.,  1916  (18),  151. 

"  Zeit.  exp.  Path.,  1910  (8),  255. 

«*  Wien.  klin.  Woch.,  1913  (26),  830. 

«5  Weston,  Jour.  Med.  Res.,  1914  (30),  377;  Stein,  Zeit.  exp.  Med.,  1914  (3), 
309. 

«  Zeit.  f.  Hyg.,  1910  (67),  279. 

6^  Zeit.  Immunitat.,  1910  (5),  76. 


232  CHEMISTRY  OF  THE   IMMIMTY  REACTIONS 

passing  through  collodion  or  porcelain  filters,  and  there  are  many  who 
hold  that  the  reacting  substance  is  a  product  of  tissue  disintegration. 
Wassermann^^  has  found  evidence  that  the  antibody  is  derived  from 
the  lymphocytes,  at  least  in  the  spinal  fluid  of  syphilitics. 

Whether  true  antibodies  are  concerned  in  the  Wassermann  reaction 
is  a  question.  In  favor  of  this  view  is  the  fact  that  the  serum  of 
rabbits  immunized  with  congenital  syphilis  livers  contains  an  anti- 
body giving  the  Wassermann  reaction,  exactly  like  the  serum  of 
syphilitics.'^^  On  the  other  hand,  the  actual  substance  of  pure  cultures 
of  spirochetes  does  not  ordinarily  act  as  antigen  with  syphilitic  sera 
in  the  Wassermann  reaction  (Noguchi).  It  is  highly  probable  that 
wdien  syphilitic  liver  extracts  are  used  as  antigen  in  the  Wassermann 
reaction,  we  have  a  true  Bordet-Gengou  reaction  of  complement  fixa- 
tion with  the  syphiUtic  substance  present  in  this  extract,  in  addition 
to  the  reaction  which  is  accomplished  by  the  lipoids. 

Whether  the  complement  is  destroyed  by  enzymes, '^'^  or  is  inhibited 
by  anti-complement  present  in  syphilitic  serum,  or  is  destroyed  by 
some  toxic  substance  in  the  serum^^  are  matters  still  under  discussion. 
A  favorite  interpretation  of  the  Wassermann  reaction,  which  seems  to 
harmonize  with  the  known  facts,  is  that  there  is  a  precipitation  of 
serum  globulin  by  the  lipoidal  colloids  of  the  antigen,  and  adsorption  of 
the  complement  by  this  precipitate. 

Apparently  the  globulins  of  the  serum  in  syphilis  are  altered  in 
some  specific  but  as  yet  unknown  way,  whereby  they  acquire  in  greatly 
increased  degree  the  capacity  to  form  this  adsorbent  precipitate."- 
Alterations  in  the  lipoids  also  seem  to  exist,  for  it  is  known  that  con- 
ditions that  modify  the  serum  lipoids  also  modify  the  reaction.  There 
seems  little  doubt  that  the  reaction  is  not  chemical  but  physical,  and 
the  union  of  complement  to  antibody  follows  essentially  the  laws  of 
adsorption.  Also  its  intimate  relation  to  the  precipitin  reaction  seems 
to  be  established  (Dean)."^ 

The  changes  in  cliaracter  of  the  blood  serum  in  sypliilis  are  sufficient  to  give 
not  only  immunological  but  also  frank  chemical  or  physico-chemical  manifesta- 
tions. For  example,  Bruck'^  states  that  the  precipitate  obtained  when  nitric 
acid  is  added  to  syphilitic  serum  is  more  abundant  and  of  a  characteristic  gelatin- 
ous appearance.  "Platinum  cloride  also  produces  a  heavier  precii>itate  in  syphilitic 
sera  (Brown  and  Iyengar).'''  The  globulin  responsible  for  the  Wassermann 
reaction  is  said  to  precipitate  more  readily  l)y  ammonium  sulphate  and  other  re- 

"*  Wa.ssermann  and  Lange,  Berl.  klin.  W'och..  1914  (51),  527. 

""  Citron  and  -Munk,  Deut.  med.  Woch.,  1910  (30).  15(30;  Eiken,  Zeit.  Imnuini- 
tiit.,  1915  (24).  ISS. 

■»  Manwaring.  Zeit.  f.  Immunitiil.,  MtOi)  CM,  .^09. 

"  Kiss.  (7>iV/.,  1910(4).  70:i. 

"See  Nathan,  Zeit.  Iminunitat.,  191S  (27>,  219;  Walker,  ,Iour.  Path,  Bact.,  1917 
(21),  184. 

"Lancet.  .Jan.  13,  1917. 

'<  Miinch.  med.  Wocli.,  1917  (04),  25.  See  al.<o  Smitii  and  Solomon,  Boston 
Med.  Surg.  Jour..  1917  (177),  .321, 

"'•  Indian. Jour,  Med.  Hes.,  1915  (3),  95. 


CYTOLYSIS  233 

agents."^  Accordinp;  to  von  Dungorn'^  the  heat  coagulaiion  of  syphilitic  serum  is 
prevented  by  a  smaller  relative  (juantity  of  an  alkaline  solution  of  indigo  than  is  the 
case  with  normal  serum,  a  statement  disputed  by  Flood  anrl  Fiijimoto."  Syphil- 
itic serum  also  flocculates  on  addition  of  appro])riate  colloidal  suspensions  which 
will  not  coagulate  normal  serum  (Vernes)."*  Landau"' states  that  syphilitic  seruni 
has  a  heightened  power  to  decolorize  and  clear  up  an  iodin  precipitate  jjrodueed  in 
the  serum.*" 

Among  other  reactions  observed  are  the  following : 

Klausner's  Serum  Reaction. — When  distilled  water  is  added  in  certain  propor- 
tions to  fresh  serum,  a  distinct  flocculent  precipitate  separates  out  in  a  few  hours, 
and  this  property  is  much  more  marked  in  syphilitic  than  in  normal  sera.  While 
not  specific  for  syphilis,  this  reaction  is  almost  invariably  present  in  certain  stages 
of  syphilis.  This  property  is  not  due  to  the  excess  of  globulin  present  in  syphilitic 
sera,  according  to  the  later  studies  of  Klausner,*'  ^vho  believes  that  the  high  lipoid 
content  of  syphilitic  serum  is  responsible. 

Porges -Hermann -Perutz  Reaction. — If  equal  parts  of  a  2%  solution  of  sodium 
gh'cocholate  and  an  alcoholic  cholesterol  suspension  (0.4%)  are  added  to  inac- 
tivated serum  from  syphilitic  patients,  a  precipitate  forms,  while  with  normal 
serum  there  occurs  no  precipitate.*-  Little  is  known  concerning  the  nature  of  this 
reaction. 

Coagulation  Reaction. — This  was  described  by  Hirschfeld  and  Klinger,*^ 
and  depends  on  the  fact  that  tissue  extracts  digested  with  syphilitic  serum  lose  their 
ability  to  coagulate  blood.  The  effect  is  believed  to  depend  on  adsorption  of  the 
lipoids  of  the  tissue  extract  by  serum  constituents,  and  hence  is  fundamentally 
similar  to  the  Wassermann  reaction. 

CYTOLYSIS  IN  GENERAL** 

Not  the  same  degree  of  success  has  been  obtained  in  immunizing 
against  other  tissue  elements  as  with  the  erythrocytes.  Immune 
serum  can  readily  be  obtained  against  cells  that  can  be  secured  quite 
free  from  other  cells,  such  as  spermatozoa,  ciliated  epithelium,  and 
leucocytes,  but  even  then  the  immunity  is  not  specific.  Much  less  is 
it  specific  when  ground-up  organs  are  used  for  immunizing,  as  is  the 
case  in  the  experimental  production  of  nephrolysins,  hepatolysins, 
etc.,  for  at  the  same  time  antibodies  are  secured  for  not  only  the 
typical  parenchyma  cells,  but  also  for  endothelium,  stroma  cells,  red 
and  white  corpuscles,  and  blood  plasma.  As  a  consequence,  the  early 
expectations  that  by  this  process  of  immunization  against  specific  cells 
great  progress  could  be  made  in  our  knowledge  of  physiology,  by 

'«  See  Heller,  Biochem.  Zeit.,  1918  (90),  166;  McDonagh.,  Proc.  Royal  Soc. 
Med.,  1916  (9),  191,  (Derm.  Sect). 

"'  Mtinch.  med.  Woch.,  1915  (62),  1212.  See  also  Flood,  Jour.  Immunol., 
1916  (2),  69;  Fujimoto,  ibid.,  1918  (3),  11. 

"*  Compt.  Rend.  Acad.  Sci.,  1918  (167),  383. 

■s  Wien.  klin.  Woch.,  1913  (26),  1702. 

*°  Not  corroborated  by  Stillians  and  Kolmer,  Jour.  Amer.  Med.  Assoc,  1915 
(64),  1459. 

«i  Biochem.  Zeit.,  1912  (47),  36. 

«2  See  Gammeltoft,  Deut.  med.  Woch.,  1912  (38),  1934;  EUermann,  ibid.,  1913 
(39),  219. 

*^  Deut.  med.  Woch.,  1914  (40),  1607.  See  also  Kolmer  and  Toyama,  Amer. 
Jour.  Syphilis,  1918  (2),  505. 

**  Literature  is  given  by  Fleischmann  and  Davidsohn,  Folia  Serologica,  1908  (1), 
173;  Landsteiner,  Handbuch  d.  Biochem.,  1909  (II  (1)  ),  542;  Ritchie,  Jour. 
Pathol,  and  Bact.,  1908  (12),  140. 


234  CHEMISTRY  OF  THE  IMMUNITY  REACTIONS 

selectively  throwing  out  of  function  an  organ  through  the  simple 
process  of  injecting  an  antiserum,  have  been  disappointed.  Equally 
little  progress  has  been  made  in  the  treatment  of  malignant  growths 
by  the  same  method.  The  immune  serums  usually  obtained  do,  to 
a  certain  extent,  injure  the  specific  organ,  but  they  also  usually 
injure  other  organs  nearly  as  much  or  perhaps  more;  furthermore  they 
generally  contain  hemolytic  toxins,  even  if  the  tissues  used  in  im- 
munizing are  free  from  blood,  and,  as  we  have  seen,  hemolytic  poisons 
may  cause  serious  tissue  destruction.^^ 

Beebe^*^  claims  to  have  secured  serums  by  immunizing  with  tissue 
nucleoproteins,  that  were  altogether  specifically  toxic  for  the  type  of 
cells  from  which  the  nucleoproteins  were  obtained;  e.  g.,  immunizing 
with  liver  nucleoproteins  yielded  serum  destroying  liver  cells  and 
no  others.  Other  observers  have  failed  to  corroborate  this  work.^^ 
According  to  Zinsser^^  the  cytolytic  antibodies  may  be  quite  distinct 
from  the  proteolytic  amboceptors  which  are  developed  against  un- 
formed protein  antigens. 

In  view  of  the  present  uncertain  state  of  the  subject,  and  the  very 
questionable  value  of  much  of  the  work  so  far  done,  the  consideration 
of  the  various  cytolysins  or  cytotoxins  may  be  dismissed  by  briefly 
referring  to  a  few  of  the  most  important  results. 

Leucocytolytic  Serum.*' — This  may  be  obtained  either  bj^  immunizing 
with  leucocytes  obtained  from  exudates  or  from  the  blood,  or  by  using  emulsions 
of  lymph-glands.  Specific  leucocytolytic  serum  agglutinates  leucocytes  and 
produces  observable  morphologic  changes,  in  the  way  of  solution  of  the  cj'toplasm 
and  cessation  of  ameboid  movements;  but  it  may  also  react  with  the  fixed  tissue 
cells  of  the  same  animal.^'*  Of  the  leucocj^tes,  the  large  granular  cells  seem  most 
affected  and  the  lymphocytes  least.  When  injected  into  the  peritoneal  cavity 
such  serum  causes  an  apparent  initial  leucopenia,  and  later  a  decided  leucocj'tosis 
in  the  peritoneal  fluid.  Corresponding  with  this,  if  bacteria  are  injected  at  the 
same  time  as  the  serum,  resistance  is  found  decreased,  but  later  it  is  much  increased. 
Such  serum  also  contains  anticomplement,  according  to  Wassermann,  indicating 
that  the  injected  leucocytes  contain  complement.  Leucocj^totoxin  obtained  by- 
immunizing  against  lymphatic  tissue  is  very  thermolabile,  being  destroyed  by  55°  C. 
for  thirty  minutes,  and  the  serum  can  be  onlj^  partially  reactivated  by  the  use  of 
fresh  serum.  Bacterial  filtrates  may  also  contain  "leucocidins"  analogous  to 
hemolysins.  Normal  foreign  sera  are  more  or  less  toxic  to  leucocytes,  which  can 
be  shown  by  the  reduced  capacity  of  the  leucocj'^tes  for  phagocytosis.*' 

Antiplatelet  Serum. — Several  experimenters  have  produced  antisera  for  plate- 
lets. Lee  and  Robertson'^  obtained  a  specific  lytic  and  agglutinative  action,  re- 
quiring complement  for  its  accomplishment.  Injected  into  animals  this  anti- 
platelet serum  caused  a  condition  resembling  exactly  purpura  hemorrhagica  in  man. 

"  See  Sata,  Ziegler's  Beitr.,  1906  (39),  1. 

»«  Jour.  Exp.  Med.,  1905  (7),  733. 

*'  Pearce  and  Jackson,  Jour.  Infect.  Dis.,  1906  (3),  742.  See  also  review  by 
.  Wells,  Zeit.  f.  Immunitat.,  1913  (19),  599. 

8«  Biochem.  Zeit.,  1916  (77),  129. 

«» Literature,  see  Flexner,  Univ.  of  Penn.  Med.  Bull.,  1902  (15),  287;  Ricketts, 
Trans.  Chicago  Path.  Soc,  1902  (5),  178;  Christian,  Deut.  Arch.  klin.  Med.,  1904 
(80),  333;  Le.schke,  Zeit.  Immunitat.,  1913  (16),  627;  Keeser,  Folia  Mikrobiol., 
1914,  H.  3. 

•"»  Spat,  Zeit.  Immunitiit.,  1914  (21),  565. 

»'  L<)hncr,  Arch.  ges.  Physiol.,  1915  (162),  129. 

»='  Jour.  Med.  Res.,  1916  (33),  323. 


CYTOLYTIC  SERA  235 

Endotheliolytic  Serum.--Every  attempt  at  imniuniziiiK  an  animal  with  any  sort 
of  fixed  tissue  must  of  necessity  involve  the  injection  of  endothelial  cells  as  well  as 
the  cells  specific  to  the  tissue  studied.  Therefore,  it  is  possible  that  cytotoxic 
serum  so  obtained  will  contain  endothelial  toxins,  and  so  complicate  any  results  of 
intra  vilam  experiments.  There  is  every  reason  to  believe  that  endotheliolytic 
substances  are  produced  in  this  way.  Ricketts ""  found  that  serum  of  animals 
immunized  against  lymph-glands  was  toxic  to  endothelial  cells,  which  was  indicated 
by  hemorrhages  at  the  point  of  injection,  and  marked  desquamation  of  endothelium 
when  the  injection  was  made  into  a  serous  cavity.  In  snake-venom  poisoning  the 
extensive  hemorrhages  are  also  due  to  an  endotheliolytic  principle,  called  by  Flexner 
hc7nnrrhagin. 

Lymphatolytic  Serum. — This  serum  has  been  studied  by  Ricketts  and  by  Flex- 
ner, who  immunized  animals  with  lymph-glands.  As  might  be  expected  from  the 
structure  of  the  injected  glands,  the  resulting  serum  contained  endotheliotoxin, 
leucocytotoxin,  hemolysin,  hemagglutinin,  leucocyto-agglutinin,  and  precipitins. 
When  injected  into  animals,  this  serum  has  a  marked  effect  upon  the  spleen  and 
lymph-glands,  producing  great  enlargement  and  congestion  of  these  structures. 
The  bone-marrow  is  also  somewhat  affected,  and  when  marrow  is  used  in  immuniz- 
ing, the  myelotoxic  serum  produces  marked  proliferative  changes  in  the  lymph- 
glands  as  well  as  in  the  marrow. 

Nephrolytic  Serum. — It  has  been  claimed  that  if  a  kidney  is  destroyed  by  li- 
gating  its  vessels  or  ureter,  the  remaining  kidney  develops  serious  degenerative 
changes,  which  are  not  present  if  one  kidney  is  entirely  removed.  This  has  been 
attributed  to  the  development  of  nephrotoxic  substances  produced  in  reaction  to 
the  absorption  of  the  injured  renal  tissue  that  has  been  left  in  the  body.  Other 
methods  of  renal  injury  have  been  thought  to  produce  similar  effects,  and  serum  of 
animals  with  kidney  disease  was  said  to  injure  the  kidneys  of  normal  animals. 
Upon  this  basis  it  has  been  thought  possible  to  explain  the  progressive  nature  of 
the  chronic  nephritides  as  the  result  of  nephrotoxins  produced  through  the  ab- 
sorption of  the  injured  cells,  which  nephrotoxins  injure  still  other  renal  cells. ^^ 
Such  a  process,  however,  involves  the  production  of  cell  toxins  in  an  animal  that 
are  toxic  for  its  own  cells,  that  is,  autocytotoxins;  and  as  it  has  so  far  been  extremely 
difficult  to  produce  autolysins  of  other  sorts,  it  is  not  altogether  probable  that  the 
kidney  is  an  exception.  Furthermore,  Pearce^*  was  unable  to  produce  isonephro- 
toxins,  and  could  not  corroborate  the  statements  as  to  the  changes  said  to  have 
been  found  in  the  remaining  kidney  after  ligating  the  vessels  of  its  mate.  He 
did  obtain  an  active  heteronephrolysin,  but  also  found  that  immunization  with 
liver  produced  nearly  as  actively  nephrolytic  serum  as  did  immunization  with 
kidney. 

Neurolytic  Serum. — Even  as  highly  specialized  cells  as  those  of  the  nervous 
tissue  seem  to  produce  a  reaction  with  the  formation  of  immune  bodies.  Perhaps 
the  most  positive  results  are  those  of  Ricketts  and  Rothstein,^^  who  found  that 
serum  of  rabbits  immunized  against  the  brains  or  cords  of  guinea-pigs  w'as  highly 
toxic  when  injected  into  the  vessels  of  guinea-pigs,  causing  death  with  various 
symptoms  only  explainable  on  the  assumption  of  nervous  lesions.  Microscopi- 
cally, the  ganglion  cells  showed  marked  changes  in  those  animals  that  survived 
the  injection  long  enough.  All  the  results  so  far  obtained  have  been  with  hetero- 
geneous serum. 3s  Venoms,  particularly  that  of  cobra,  possess  strong  neurolytic 
substances  that  are  the  chief  toxic  agents  in  most  of  the  venoms  (rattlesnake  venom 
excepted). 

Thy  rely  tic  Serurh. — There  are  but  few  reports  on  this  serum,  but  that  of 
Portis^'  indicates  that  after  removal  of  all  hemolysis  as  a  factor  there  do  occur 
changes,  in  the  nature  of  excessive  absorption  of  colloid,  and  proliferation  after 
the  order  of  that  seen  in  thyroid  regeneration.  However,  the  clinical  pictvire  of 
thryoidectomy  was  not  produced  in  any  case,  and  the  anatomic  changes  w'ere 
not  great.     By  immunizing  against  nucleoproteins  derived  from  thyroid  tissue, 

"  See  Kapsenberg,  Zeit.  Immunitat.,  1912  (12),  477. 
"  Univ.  of  Penn.  Med.  Bull.,  1903  (16),  217. 
55  Trans.  Chicago  Path.  Soc,  1903  (5),  207. 

'^  An  attempt  to  obtain  a  specific  neurotoxin  with  corpus  striatum  was  un- 
successful.    (Lillian  Moore,  Jour.  Immunol.,  1916  (1),  525.) 
"  Jour.  Infectious  Diseases,  1904  (1),  127. 


236  CHEMISTRY  OF  THE  IMMCMTY  REACTIONS 

Beebe^*  has  secured  an  antiserum  to  wliich  he  ascribes  some  effect  upon  diseased 
thyroids  (exophthalmic  goiter).  MacCallum^'  could  not  get  a  specific  semm  for 
parathyroid  tissue. 

Numerous  reports  may  be  found  indicating  attempts,  with  varying 
success,  to  obtain  serum  toxic  for  other  tissues.  Among  them  may  be 
mentioned  epitheliolysi7i^  (for  ciliated  epitheHum),  spermatotoxin,'^ 
hepatolysin,  cardiolysin,  splenolysin,  and  syncytiolysin.^  Special  at- 
tention has  been  given  to  the  production  of  specific  lysins  for  cancer 
cells,  without  definite  success.  (See  Chapter  xix.)  In  general  it 
can  be  said  that  it  has  7iot  been  found  possible  in  this  way  to  throw 
out  of  function  one  particular  organ,  with  or  without  involvement  of 
other  structures. "*  The  principles  involved  in  all  these  experiments  are 
the  same,  and  the  results  are  in  no  instance  altogether  satisfactory; 
therefore  no  further  consideration  of  these  special  cytotoxic  serums 
will  be  made  here,  the  reader  being  referred  to  other  sources  for  de- 
tails.^ It  may  be  said,  however,  that  recent  developments  indicate 
that  various  tissues  not  only  contain  proteins  which  exhibit  the  species 
characteristics  of  the  entire  animal,  but  also  other  proteins  or  anti- 
genic radicals  which  are  more  or  less  independent  of  these  and  char- 
acteristic to  a  certain  degree  for  the  tissue  from  which  the  antigen 
was  obtained.  This  being  the  case,  we  cannot  consider  the  problem 
of  specific  cytotoxins  a  closed  chapter;  improved  methods  for  sepa- 
rating our  antigens  may  yet  enable  us  to  secure  antibodies  specific  for 
a  single  tissue  or  organ.  (See  Specificity  of  Antigens,  Chap,  vii.) 
However,  by  the  useful  method  of  studying  the  eifect  of  cytotoxic 
serum  on  the  growth  of  tissue  cultures  in  vitro,  Lambert^  found  no 
evidence  whatever  of  specificity,  although  there- is  a  non-specific  inhi- 
bition of  growth  by  the  immune  sera. 

«»  Jour.  Amer.  Med.  Assoc,  1906  (46),  484.       Not  corroborated  by  Portis  and 
Bach,  ibid.,  1914  (62),  1884. 
3«  Med.  News,  1903  (83),  820. 

1  See  Galli-Valerio,  Zeit.  Immunitilt,  1915  (24),  311. 

^Taylor  (Jour.  Biol.  Cheni.,  1908  (5),  311)  made  the  interesting  observation 
that  no  spermatolytic  serum  could  be  obtained  by  immunizing  with  isolated 
nucleic  acid,  protamines,  or  ether  extracts  of  sperm,  although  immunizing  with 
whole  sperm  produced  active  sera. 

3  Lake,  Jour.  Infect.  Dis.,  1914  (14),  385. 

^  .\n  attempt  to  produce  a  specific  cytolytic  serum  for  tlie  islands  of  Langerhans 
by  Kamimura  was  unsuccessful.  (Mitt.  med.  Fak.  Univ.  Tokio,  1917  (17),  95). 
Ogata,  however,  reports  the  production  of  a  specific  IhymoCoxic  serum  (Rep.  Univ. 
Kioto,  1917  (1),  449). 

**  Biochemisches  Centralblatt,  1903  (1),  573,  el  scq.;  also  see  Sata,  Ziegler's 
Beitr.,  1906  (39),  1;  and  literature  cited  previously. 

«  Jour.  Exp.  Med.,  1914  (19),  277;  also  Walton,  ibid.,  1915  (22),  194. 


CHAPTER  X 

CHEMICAL   MEANS   OF  DEFENSE  AGAINST  NON- 
ANTIGENIC   POISONS' 

Although  the  examples  of  acquired  imniunity  against  poisons  of 
known  chemical  composition  arc  few  indeed,  nevertheless  the  body 
possesses  means  of  defense  against  many  such  poisons,  which  decrease 
to  greater  or  less  degree  their  harmful  effects.  It  is  to  be  noted,  how- 
ever, that  the  increased  tolerance  to  such  poisons  is  far  less  than  the 
degree  of  tolerance  characteristic  of  innnunity  to  true  toxins;  thus,  in 
arsenic  eaters  the  maximum  observed  tolerance  is  but  three  or  four 
times  the  minimum,  and  less  than  the  certainly  fatal  dose  (Hausmann) ; 
dogs  can  be  made  tolerant  to  only  iibout  three  times  the  fatal  dose  of 
morphine  (Faust).  Furthermore,  with  many  poisons  of  this  class  the 
tolerance  is  largely  fictitious,  since  in  spite  of  the  absence  of  acute 
svmptoms  chronic  poisoning  is  taking  place;  and,  of  course,  with  many 
poisons  no  distinct  increase  of  tolerance  can  be  produced.  True 
immuniiy,  associated  with  the  production  of  neutralizing  substances 
in  the  blcod,  has  as  yet  been  obtained  only  against  substances  of  pro- 
tein nature  or  substances  very  closely  resembling  Ihe  proteins.  Ehr- 
lich-  believed  that  simple  toxic  chemicals  are,  like  toxins,  bound  to  the 
cells  by  special  receptors,  chemoreceptors,  which,  in  view  of  their  simpler 
function  may  be  assumed  to  be  simpler  than  the  receptors  for  toxins. 
They  seem  to  be  more  firmly  fixed  to  the  cells,  and  being,  therefore, 
less  easily  discharged  than  bacterial  receptors  no  free  antibodies  are 
produced  bj^  immunization.  To  be  sure,  there  have  been  observations 
interpreted  as  evidence  of  imnumity  to  large  molecular  complexes, 
especially  such  as  lipoids  and  glucosides,  but  as  yet  the  positive  estab- 
lishment of  the  formation  of  antibodies  by  reaction  to  non-protein 
antigens  has  not  been  accomplished.  It  must  be  taken  into  considera- 
tion, however,  that  various  chemical  substances  introduced  into  the 
blood  or  tissues  of  an  animal,  may  form  compounds  with  the  animal's 
proteins  which  behave  like  foreign  proteins,  to  which  the  animal 
reacts  by  becoming  hypersensitive;  in  this  way  are  explained  the 
instances  of  idiosyncrasy,  with  reactions  of  anaphylactic  character, 
which  are  sometimes  shown  with  iodoform,  antipj'rine,  salvarsan,  and 
other  substances.     (See  Antigens,  Chapter  vii.) 

Studies  on  bacterial  immunity  and  allied  topics  have  as  yet  shown 
nothing  to  explain  the  acquirement  of  tolerance  to  morphine,  alcohol 

'  Bibliography  by  Hausmiinn,  Ergebnisse  Physiol.,  1907  (6),  58. 
^  Beitrage  z.  exp.  Path.  u.  Chem.,  Leipzig,  1909,  p.  189. 

237 


238  DEFENSE  AGAINST  NON-ANTIGENIC  POISONS 

arsenic,  and  other  similar  poisons.  A  few  observers  have  claimed  that 
the  serum  of  animals  immunized  to  morphine  will  neutralize  to  some 
degree  the  toxic  effects  of  morphine,  but  these  results  have  not  been 
generally  substantiated.  Others  have  claimed  that  increased  oxida- 
tive powers  are  developed  under  the  stimulation  of  the  poison,  which 
permits  of  its  more  rapid  destruction,  especially  in  the  liver,  but  the 
experimental  support  of  this  hypothesis  is  slight.  Still  another  idea 
is  that,  at  least  in  the  case  of  morphine,  decomposition  products  are 
produced,  and  accumulate  in  the  body,  that  neutralize  physiologically 
to  some  extent  the  morphine  itself;  this  hypothesis  can  scarcel}''  be 
applied  to  arsenic  and  alcohol  tolerance.^  It  has  been  found  that 
in  animals  habituated  to  morphine  there  is  an  increased  power  to  de- 
stroy morphine,  but,  nevertheless,  the  blood  of  such  animals  still  con- 
tains quantities  of  morphine  toxic  for  normal  animals,  so  there  must 
be  a  certain  refractoriness  or  cellular  immunity  in  addition  (Riib- 
samen).  Schweisheimer*  has  shown  that  when  chronic  alcohoUcs 
and  total  abstainers  are  given  equal  quantities  of  alcohol,  the  alcohol 
content  of  the  blood  reaches  a  higher  level,  and  persists  for  a  longer 
time  at  a  high  level,  in  the  abstainers.  Apparently  the  alcohol-habit- 
uated organism  can  destroy  alcohol  more  readily,  presumably 
through  more  rapid  oxidation.^  However,  other  factors  are  involved 
in  alcohol  tolerance,  for  with  equal  quantities  of  alcohol  in  the  blood 
the  abstainers  show  a  more  marked  intoxication  than  the  habitual 
drinker.  So,  too,  in  morphine  tolerance  any  general  resistance  through 
augmented  oxidation  seems  inadequate  in  view  of  the  specific  increase 
in  the  tolerance  of  the  respiratory  center  observed  in  this  condition.^ 
Also  we  find  that  tolerance  to  one  drug  may  be  accompanied  by  toler- 
ance to  other  drugs  exerting  similar  physiological  action.'' 

It  is  possible,  also,  that  the  cell  constituents  with  which  the  poisons 
ordinarily  combine  are  produced  in  increased  amounts  under  the 

'  Concerning  immunity  against  morphine  see  DuMez,  Jour.  Amer.  Med.  Assoc, 
1919  (72),  1069;  full  bibliography.  He  summarizes  the  evidence  as  follows: 
"The  only  knowledge  of  a  positive  nature  that  we  have  at  present  concerning  these 
problems  is  that  the  different  organs  and  centers  of  the  body  acquire  tolerance  to 
morphine  and  heroine  to  a  different  degree  and  with  varied  degrees  of  readiness; 
that  these  drugs  as  such  are  excreted  in  the  feces  in  diminishing  amounts  during 
the  period  of  acquiring  tolerance;  and  that  there  is  evidently  present  in  the  blood 
serum  of  tolerant  animals  (dogs)  during  periods  of  abstinence  a  substance  or  sub- 
stances which,  when  injected  into  normal  animals  of  the  same  species,  causes  the 
appearance  of  symptoms  identical  with  the  so-called  withdrawal  phenomena. 
Whether  or  not  the  disappearance  of  these  drugs  from  the  feces  is  due  to  their 
increased  destruction  in  the  organism  is  still  an  unsettled  question.  It  has  not 
been  proved  that  the  destruction  of  morphine  in  the  organism,  if  it  does  take 
place  to  an  increased  degree,  is  a  causative  factor  in  the  production  of  tolerance. 
It  may  be  only  a  concomitant  phenomenon." 

*  Deut.  Arch.  klin.  Med.,  1913  (109),  271. 

6  See  also  Voltz  and  Dietrich,  Hiochciu.  Zcit,  IDl")  (08),  118.  J.  Hirsch, 
ibid.,  191()  (77),  129. 

6  Vtui  Dongcn,  Arch.  g(>s.  Physiol.,  1915  (lt)2),  54. 

^  So(!  Myers,  jour.  I'harmacol..  1910  (8),  417.  lIowevcM-,  Biberfeld  finds  mor- 
phine tolerance  to  be  specific  (Biocnem.  Zcit.,  1910  (77),  283). 


TOLERANCE  TO  POISONS  239 

stimulus  of  the  poison,  just  as  they  are  in  the  case  of  immunization 
with  toxins,  with  the  difference  that  the  combining  substances  are 
not  thrown  off  into  the  blood.  For  example,  it  has  been  claimed  that 
arsenic  is  ordinarily  combined  and  held  in  the  liver  by  a  nuclcoprotein, 
and  the  suggestion  has  been  made  that  in  arsenic  habituds  this  nuclco- 
protein is  increased  in  amount.  Again,  saponin  seems  to  act  upon  the 
cholesterol  of  the  red  corpuscles,  and  Kobert  observed  increased  resist- 
ance to  the  action  of  saponin  exhibited  by  the  serum  of  immunized 
animals,  which  he  attributes  to  an  increased  amount  of  cholesterol, 
perhaiis  liberated  by  the  corpuscles  decomposed  by  the  injected  poison, 
or  perhaps  produced  in  excess  by  the  tissues.  Wohlgemuth^  has  also 
suggested  that  in  the  case  of  poisoning  with  large  amounts  of  sub- 
stances which  combine  with  glycuronic  acid  (e.  g.,  lysol),  excessive 
quantities  of  this  substance  are  formed  by  the  cells  and  excreted  into 
the  blood,  where  they  neutralize  the  poisons  in  much  the  same  manner 
as  the  antitoxins  neutralize  toxins. 

But  besides  these  scanty  examples  of  tolerance  to  poisons,  the  body 
possesses  a  number  of  methods  for  opposing  manj'-  other  poisons  with 
more  or  less  success;  and,  poisons  invariably  acting  chemically,  the 
defenses  are  in  turn  largely  chemical.  We  have  elsewhere  referred  to 
the  destructive  action  of  the  enzymes  of  the  digestive  tract  upon  bac- 
terial and  similar  poisons;  this  means  of  defense  cannot  apply  to 
non-protein  chemical  substances  except  possibly  glucosides  and  toxic 
lipoids.  But  the  acidity  of  the  gastric  juice,  the  alkalinity  of  the 
bile  and  pancreatic  juice,  and  the  precipitating  effect  of  the  hydrogen 
sulphide  formed  in  intestinal  putrefaction  are  all  factors  that  help 
to  neutralize  or  prevent  the  absorption  of  certain  poisons,  their  total 
efficiency,  however,  being  on  the  whole  very  slight.  After  absorption 
of  a  poison  a  large  series  of  chemical  reactions  and  physiological  proc- 
esses is  brought  into  play,  and  there  are  few  poisons  indeed  whose 
harmful  influence  is  not  more  or  less  decreased  by  these  means.  Ko- 
bert" classifies  these  protective  processes  as  follows: 

1.  Rapid  elimination,  either  before  absorption  by  means  of  diarrhea  and 
vomiting,  or  by  the  same  means  after  absorption  in  case  the  poisons  are  excreted 
into  the  digestive  tract  (e.  g.,  morphine,  venoms,  antimony,  and  many  other 
metals).  Many  poisons  are  very  rapidly  eliminated  by  other  routes  (e.  g.,  anes- 
thetics, curare),  in  some  instances  causing  harm,  particularly  to  the  eliminating 
organ  (e.  g.,  kidneys  in  phenol  poisoning,  intestines  in  ricin  poisoning).  The 
routes  and  conditions  of  elimination  of  poisons  have  been  fully  discussed  by 
Lewin." 

2.  Deposition  and  Fixation  in  Single  Organs  or  Tissues. — In  this  respect  the 
liver  is  especially  important,  probably  because  of  its  location  and  function  as  a 
filter  for  all  the  blood  coming  fresh  from  the  alimentary  tract."     The  manner  and 

»  Biochem.  Zeitschr.,  1906  (1),  134. 

^  "Lehrbuch  der  Intoxikationen,"  Stuttgart. 

10  Deut.  med.  Woch.,  1906  (32),  169;  see  also  Mendel  et  al.,  Amer.  Jour.  Physiol., 
1904  (11),  5;  1906  (16),  147  and  152. 

"  Concerning  the  detoxicating  function  of  the  liver  see  Woronzow,  Dissertation, 
Dorpat,  1910;  Rothberger  and  Winterberg,  Arch,  internat.  Pharmacodyn.,  1905 
(15),  339. 


240  DEFENSE  AGAINST  NON-ANTIGENIC  POISONS 

means  by  which  this  fixation  is  brought  about  are  unknown.  It  is  possible  that  the 
power  of  the  tissues  to  bind  poisons  may  become  increased  bj'  repeated  doses,  lead- 
ing to  "specific  acquired  tolerance. '"^^  According  to  Slowtzoff^' arsenic  is  fixed 
by  the  nucleus  in  a  very  firm  combination;'*  mercury  by  globulins  in  a  less  stable 
combination;  copper  by  the  nucleins,  but  less  firmly  than  the  arsenic.  Other 
poisons,  chiefly  alkaloids,  are  probably  combined  with  bile  acids.  Possibly  some 
poisons  combine  with  glycogen.  These  compounds  are  but  slowly  broken  up,  and 
thus  the  poison  reaches  the  more  susceptible  and  more  important  tissues  in  a  rela- 
tively diluted  condition.  The  bones  seem  to  hold  in  harmless  form  poisonous 
fluorides,  and  to  less  extent  arsenic,  barium,  and  tungsten,  which  persist  in  the 
bones  for  a  great  length  of  time.  Leucocytes  are  possibly  important  binders  of 
poisons,  perhaps  through  combination  with  their  nucleins,'^  but  storage  in  these 
labile  cells  is  necessarily  of  relatively  brief  duration. 

3.  Combination  with  substances  formed  or  contained  in  the  tissues;  the  result- 
ing substance  being  less  toxic  than  the  poison  alone.  Under  this  heading  may  be 
included  both  chemical  combination  and  physical  absorption  or  solution,  such  as 
the  deviation  of  the  lipoid-soluble  narcotics  from  the  central  nervous  system  by 
excessive  tissue  fats,  or  by  fats  therapeutically  introduced.'*  Many  poisons 
combine  with  the  inorganic  constituents  of  the  tissues;  e.  g.,  btyium  and  various 
aromatic  substances  with  SO4;  silver  with  CI,  etc. 

4.  Chemical  alteration,  with  or  without  subsequent  combination  with  other  sub- 
stances, by  such  means  as  oxidation,  reduction,  hydrolysis,  and  neutralization. 

5.  Impaired  absorption  should  also  be  considered  as  a  means  of  defense  against 
poisons.  This  may  depend  upon  the  injury  to  the  gastro-intestinal  tract  produced 
either  by  the  poison  itself  or  by  some  independent  pathological  condition.  Cloetta 
considers  impaired  absorption  important  in  acquired  immunity  to  arsenic  (see 
below)  and  it  may  also  modify  the  effects  of  other  poisons." 

The  chemical  reactions  employed  in  defense  against  simple  chemical 
poisons  have  been  particularly  considered  bj^  E.  Fromm,^^  whose  out- 
line is  here  partially  followed,  and  to  which  the  reader  is  referred  for 
bibliography. 

INORGANIC  POISONS 

MetaUic  poisons,  such  as  lead,  silver,  mercury,  and  arsenic,  are 
made  insoluble,  particularly  by  forming  compounds  with  proteins  in 
the  alimentary  tract,  intestinal  walls,  blood,  or  internal  organs;  also 
by  forming  sulphides  with  the  H2S  of  the  intestinal  contents.  Accord- 
ing to  Cloetta^^  immunization  against  arsenic  depends  entirely  upon 
a  reduction  of  absorption  in  the  intestine,  for  the  longer  arsenic  is 
taken,  the  less  appears  in  the  urine  and  the  more  appears  in  the  feces. ^^ 
At  the  same  time  the  resistance  to  arsenic  injected  subcutaneouslj^ 
is  not  increased  at  all,  and  no  increase  in  resistance  can  be  obtained 

'2  Santesson,  Skand.  Arch.  Physiol.,  1911  (25),  28. 

'■'  Hofmeister's  Beitr.,  1901  (1),  281;  1902  (2),  307. 

"  Denied  by  Heff'ter.  (Arch,  intcrnat.  de  Pharmacodyn.,  1905  (15),  399),  who 
considers  it  more  a  i)hysico-clK'mical  process. 

'*  Stessano,  Comjjt.  Rend.  Acad.  Sci.,  1900  (131),  72. 

'«See  (Iraham,  Jour.  Inf.  Dis.,  1911  (8),  147. 

"v.  Lhota,  .Vrch.  internal,  pharmacodyn.,  1912  (22),  (il. 

'*  "Die  cheniischen  Schiitzmittel  des  Tierkorpers  bei  Vergiftungen,"  Strassburg, 
Karl  Triibner,  1903.  See  also  r6sum6  by  EUinger,  Deut.  med.  Woch.,  1900  (26), 
580. 

'*  Arch.  exp.  Path.  u.  Pharm.,  1906  (54),  196;  Corresponbl.  Schweizer  .\oTzte, 
1911  (41).  737. 

^"  Not  accepted  bv  lluusmann,  Krgel)nisse  Physiol.,  1907  (6),  58;  or  Joachi- 
nioulu,  .Vrch.  cxi).   Path..   1916  (79).  119. 


DEFENSE  AOAIXST  ISOliGANIC  I'OISOSS  241 

by  repeated  siihcutaiicous  injecdoiis  of  sul)letluil  doses,  'riicic  is, 
however,  reason  to  question  the  authenticity  of  the  reputed  toku-ance 
of  habituds  to  arsenic  (Joachimoglu).-"  Antimony  does  not  produce 
tolerance  in  experimental  animals  (Cloetta).-'  The  manner  in  which 
various  inorganic  ions  antagonize  the  physiological  action  of  one 
another  (e.  g.,  sodium  and  potassium,  calcium  and  nuignesium)  is 
still  an  important  problem. -'- 

Free  acids  and  alkalies  are  partly  neutralized  by  the  alkaline  and 
acid  contents  of  the  gastro-intestinal  tract,  partly  by  forming  com- 
pounds with  the  proteins,  and  partly  by  the  alkalies  and  carbonic  acid 
of  the  blood  stream.  (See  "Acid  Intoxication,"  Chap,  xx.)  Phos- 
phorus^^  and  sulphides  arc  oxidized  after  absorption  into  phosphoric 
and  sulphuric  acid,  which  are  in  turn  neutralized  by  the  alkalinity 
of  the  blood^and  tissues.  Lillie-^  has  called  attention  to  the  close, 
palisade  arrangement  of  the  nuclei  of  the  epithelium  lining  the  ali- 
mentary tract,  which  makes  it  necessary  for  all  substances  absorbed 
to  pass  through  the  zone  of  their  active  oxidative  influence,  a  fact 
uncloubtedly  of  great  importance  in  the  defense  of  the  body. 

Reduction  of  iodic  acid,  chloric  acid,  hj-pochlorous  acid,  and  their 
salts  occurs  in  the  body,  resulting  in  their  conversion  into  the  much 
less  toxic  iodides  and  chlorides.  Tellurium  compounds  are  also  re- 
duced and  rendered  insoluble.  This  reaction  occurs  to  some  extent 
in  the  intestines;  how  much  in  other  organs  is  unknown. 

Methylation,  the  addition  of  CH3  groups,  is  observed  in  poisoning 
by  tellurium,  which  is  eliminated  in  the  breath  as  methyl  telluride, 
and  also  in  the  sweat  and  feces. ^^  Selenium,  pyridine,  and  some  other 
substances  also  combine  with  methane.  The  source  of  the  methane  is 
possibly  in  the  xanthine  molecule. 

Summary. — There  are,  therefore,  three  chief  reactions  used  against 
inorganic  poisons  in  the  body,  oxidation,  reduction,  and  splitting  off 
of  water;  neutralization  of  acids  or  alkalies  and  the  formation  of  al- 
buminates and  sulphides  being  included  under  the  last  heading,  since 
in  these  reactions  the  splitting  off  of  water  is  an  essential  step. 

ORGANIC  POISONS 

In  the  case  of  organic  poisons  an  ecjually  small  number  of  primary 
reactions  is  emploj-ed  in  their  detoxication,  but  in  more  complicated 
manners  and  combinations  corresponding  with  the  complexity  of 
organic  compounds. 

-'  Arch.  exp.  Path.  u.  Phann.,  1911  (64),  352. 

•"  See  Osterhout,  Proc.  Phil.  Soc,  1916  (55),  533. 

-'  Increased  tolerance  to  phosphorus  maj'  be  obtained  by  repeated  small  doses, 
but  it  lasts  only  while  the  poison  is  being  given  continuously  (Oppel,  Ziegler's 
Beitr.,  1910  (49),  543).  Accompanying  the  tolerance  are  structural  changes  in 
the  liver  cells  to  which  are  ascribed  some  significance  bv  Oppel. 

-*  Amer.  Jour.  Physiol..  1902  (7),  412. 

"  See  Mead  and  Gies,  Amer.  Jour.  Phvsiol.,  1901  (5),  105.  Caffein  may  be 
demethylated  in  the  liver.  Kotake,  Zeit.,  physiol.  Chem.,  1908  (57),  378. 

16 


242  DEFENSE  AGAINST  NON-ANTIGENIC  POISONS 

Oxidation,  which  has  abeady  been  mentioned  as  a  means  of  de- 
struction of  bacterial  toxins,  is  naturally  one  of  the  most  effective 
agents  in  the  destruction  of  simpler  organic  substances,  since  the 
ordinary  decomposition  of  all  organic  food-stuffs  is  through  oxidation. 
There  are  numbers  of  specific  examples  of  the  conversion  of  a  poisonous 
into  a  less  poisonous  or  non-poisonous  substance  by  oxidation.  All 
acids  of  the  fatty  acid  series  are  oxidized  vigorously  in  the  body, 
eventually  into  CO2  and  H2O;  and  pathologically  produced  acetic 
and  lactic  acids  are  destroyed  in  this  way.  The  hver  contains  an 
oxidase  destroying  alcohol,  which  is  not  increased  in  the  livers  of 
animals  made  tolerant  to  alcohol  (J.  Hirsch).-^  Uric  acid  is  oxidized 
vigorously  by  many  organs  (except  in  man),  as  are  other  members  of 
the  purine  series,  such  as  caffeine  and  theobromine.  Presumably  oxi- 
dation of  organic  poisons  as  well  as  of  food-stuffs  is  brought  about  by 
the  oxidizing  enzymes  of  the  cells,  as  shown  by  Ehrlich's  indophenol 
reaction,  which  consists  of  the  oxidation  of  paraphenylene  diamine 
and  a-naphthol,  with  a  resulting  synthesis.  This  reaction  is  said  by 
Lillie^'^  to  occur  principally  in  and  about  the  cell  nuclei  or  cell 
membranes. 

Combination,  with  or  without  Preliminary  Oxidation. — Oxi- 
dation is  also  an  essential  preliminary  step  to  many  of  the  protect- 
ing combinations,  in  which  a  cell  constituent  is  united  to  an  organic 
poison.     The  most  important  of  these  combining  substances  are: 

1.  Sulphuric  Acid. — One  of  the  earliest  and  most  important  observa- 
tions on  the  protective  action  of  sulphuric  acid  was  made  by  Baumann 
and  Herter,--  who  showed  that  phenol  is  eliminated  as  a  potassium 
salt  of  the  sulphuric  acid  derivative,  as  follows: 

CeHsOH  +  HO-SO3K  =  C^HsO-SOsK  +  HoO, 

a  reaction  that  has  been  put  to  practical  use  in  treating  phenol  poison- 
ing. As  phenol  and  cresols  are  produced  constantly  in  intestinal  de- 
composition, this  reaction  is  undoubtedly  of  great  service,  since  the 
salt  formed  is  relatively  harmless.  Indole  and  skatole  are  similarly  de- 
toxicated  by  being  converted  into  corresponding  salts,  but  onh'  after 
a  preliminary  oxidation  into  indoxyl  and  skatoxijl,  according  to  the 
following  reaction : 


CH 

C(OH) 

C6H4<^^CH  +  0 

=  C6H4^^CH. 

NH 

NH 

(indole) 

(indoxyl) 

C(OH)  C-O-SO2OK. 

CaH4<^^CH  +  HO— SOjOK  =C«hZ^CH  +  H2O. 

NH  NH 

(indoxyl)  (indican) 

"  Biochem.  Zeit.,  1916  (77),  129. 

"  Zdt.  physiol.  Cliem.,  1877  (1),  247. 


DEFENSE  AGAINST  ORGANIC  POISONS  243 

A  host  of  other  aromatic  organic  substances  are  similarly  combined 
with  sulphuric  acid,^*  with  or  without  preliminary  oxidation,  includ- 
ing all  substances  resembhng  phenol  or  which  through  oxidation  are 
changed  into  phenols,  such  as  cresol,  thymol,  anilin,  naphthalin,  pyro- 
gallol,  and  tannin.  By  this  means  a  poisonous  substance  is  converted 
into  a  relatively  harmless  one,  which  is  readily  soluble  and  rapidly 
eliminated. 

2.  Glycuronic  acid  occupies  the  same  position  as  sulphuric  acid,  com- 
bining particularly  with  naphthol,  thymol,  camphor,  chloral  hydrate, 
and  but}'!  chloral.  Sometimes  a  substance  may  appear  in  the  urine 
combined  in  part  with  sulphuric,  in  part  with  glycuronic  acid,  show- 
ing the  similarity  of  their  function.  Apparently  when  there  is  not 
sufficient  sulphuric  acid  in  the  body  to  combine  with  all  the  poison, 
the  excess  unites  with  glycuronic  acid,^^  although  combination  between 
glycuronic  acid  and  the  aromatic  substance  begins  to  occur  before  all 
the  sulphuric  acid  is  exhausted. ^"^  Glycuronic  acid  represents  merely 
a  first  step  in  the  oxidation  of  glucose,  as  follows : 

OHC-(CHOH)4-CH20H  +  00  =  0HC-(CH0H)4-C00H  +  H2O. 
(glucose)  (glycuronic  acid) 

This  oxidation  occurs  after  the  aldehyde  group  of  the  glucose  has 
been  combined  by  some  other  substance;  hence  the  aldehyde  group 
escapes  oxidation,  although  ordinarily  more  easily  oxidized  than  the 
alcohol  group. 

Just  as  with  the  addition  of  sulphuric  acid,  oxidation  may  be  a 
preliminary  step  to  the  addition  of  glycuronic  acid;  e.g.,  naphthalin 
is  oxidized  into  a-naphthol,  before  uniting  to  glycuronic  acid,  as  fol- 
lows: 

H    H 
/C=C\        H  H    H 

HC4  ^C-C^  /C  =  C\         OH 

^C-Cf  >CH  +  O  =  HC<  >C-C^ 

H        \C  =  C/  ^C-Cf  >CH 

H    H  H        \C  =  C/ 

H    H 
(naphthalin)  (a-naphthol) 

The  same  is  the  case  with  many  camphors  and  terpenes.  Reduction 
may  be  the  preliminary  step,  as  with  chloral  hydrate,  which  is  first 
reduced  to  trichlor-ethyl-alcohol.  In  still  other  cases  splitting  off  of 
water  is  the  chief  preliminary  step. 

3.  Glycine  is  one  of  the  longest  known  combining  substances,  the 
observation  of  the  combination  of  glycine  with  benzoic  acid  to  form 
hippuric  acid  being  the  first  proof  of  synthesis  in  the  animal  body  dis- 
covered by  Wohler  (1824).     The  reaction  is  as  follows: 

CeHsCOOH  +  H2N-CH2-COOH  =  C6H5CO-HN-CH2-COOH  +  H2O. 
(benzoic  acid)         (glycine)  (hippuric  acid) 

28  3gg  Hammarsten's  Text-book  (fourth  American  ed.).  P-  542. 

28  See  Austin  and  Barron,  Boston  Me^.  and  Surg.  Jour.,  1905  (152),  269. 
Wohlgemuth  has  observed  a  case  in  which  all  the  sulphuric  acid  of  the  urine  was 
in  organic  combination  (Berl.  klin.  Woch.,  1906  (43),  508). 

30  See  Salkowski,  Zeit.  physiol.  Chem.,  1904  (42),  230. 


244  DEFENSE  AGAINST  NON-ANTIGENIC  POISONS 

A  special  enzyme  has  Ijeen  found  in  kidney  substance  vvliicli  can  bring 
about  this  reaction  outside  the  body.  Normally  this  enzyme  occurs 
chiefly  in  the  kidney  l)ut  may  also  occur  in  other  organs.  Man}'  other 
aromatic  compounds  also  combine  with  glycine  before  elimination, 
e.  g.,  salicylic  acid.  Some  are  first  altered  to  a  suitable  form  by 
oxidation;  e.  g.,  toluene  is  oxidized  to  benzoic  acid,  xylene  to  toluic 
acid,  nitro-benzaldehyde  to  nitro-benzoic  acid.  Many  of  the  sub- 
stances that  can  be  made  to  combine  with  glycine  in  the  body  are  of 
such  a  foreign  nature  that  they  never  could  need  neutralization  under 
any  other  than  experimental  conditions,  but  here,  as  with  the  sul- 
phuric and  glycuronic  acid  reactions,  combination  occurs  whenever  a 
suitable  substance  is  present  in  the  blood,  glycine  alwa^'s  being  abun- 
dant as  a  cleavage  product  of  the  proteins. 

4.  Urea  ma}'  also  be  a  means  of  defense,  forming  salts  with  organic 
acids  which  are  rapidly  eliminated;  e.  g.,  amido-benzoic  acid  and  nitro- 
hippuric  acid. 

5.  Methane. — Methylation,  which  occurs  also  with  tellurium,  is 
observed  on  administration  of  pyridine,  as  shown  by  the  following 
equation: 

H     H  H    H 

HC/  ^N  +  CH,  +  O  =  Kcf  S^/ 

H     H  H    H 

(pyridine) 

This  reaction  is  of  special  importance,  because  many  alkaloids  contam 
a  pyridine  group;  and  the  resulting  methyl  compound  may  be  less 
toxic  than  the  original  alkaloid  — e.  g.,  methyl  morphine. 

6.  Sulphur  split  off  from  proteins  may  combine  with  CNH  and 
CNK,  converting  them  into  the  much  less  toxic  sulphocyanides.^' 

7.  Bile  Acids. — All  the  above  mentioned  reactions  are  protective 
largely  because  the  substances  formed  are  soluble  and  rapidly  elim- 
inated, as  well  as  being  less  toxic  than  the  original  poison.  Com- 
pounds of  many  poisons  are  formed  with  bile  acids  which  are  insoluble, 
and  therefore  only  slowly  dissolve  or  decompose,  thus  protecting  the 
body  from  overwhelming  doses  of  the  poison.  Such  compounds  are 
formed,  not  only  with  inorganic  poisons,  but  also  with  alkaloids,  espe- 
cially strychnine,  brucine,  and  quinine.  They  ar(^  then  tlepositetl  in 
the  livei',  to  be  slowly  dissolved  and  eliminated. 

((Occasionally  acetic  acid  and  cysteine  have  been  observed  to  act  as 
combining  substances.  Calcium  may  be  considered  a  defensive  agent 
against  certain  poisons  [oxalic  and  citric  acids]  with. which  it  forms 
insoluble*  compounds,  although  it  is  probable  that  the  toxicity  of  oxa- 
lates depends  largely  upon  their  robbing  the  cells  of  calcium.''-) 

"'  See  Meuriee,  Arch.  int.  I'liariiuicodyii.,  1900  (7),  11. 

■"' See  Robert  son  .•nil  I  Miniicl  I,  .lour.  I'harniiicol.,  IIHL'  (:>),  «);5.'). 


Mi-rriioDs  or  defksse  '1\') 

Neutralization  of  oi'^aiiic  acids  entering  llic  Ixxly  or  Ioi'iikmI  in 
iiictabolisin  is  accomplished  by  the  sodium  carbonate  of  the  blood 
when  ill  small  amounts;  if  excessive  in  quantity  {e.  g.,  diabetic  coma), 
a  portion  is  combined  with  ammonia  and  appears  as  an  ammonium 
salt  in  the  urine.  Mafjiiesium  and  calcium  salts  may  also  help  in  the 
neutralization,  prolKil)ly  at  the  expense  of  the  bone  tissue.'''  (See 
"Acid  Intoxication,"  Chap.  xx). 

Dehydration,  which  plays  a  prominent  part  in  a  numlier  of  the 
abovc-nuMitioned  syntheses,  is  particularly  important  in  the  change 
of  ammonium  carbonate  into  urea: 

NH.— Ov  NH2. 

>C0  =  >CO  +  21120 

NH4— O^  nh/ 

As  ammonium  salts  of  all  sorts  are  very  toxic,  especially  hemolytic, 
while  urea  is  not,  this  process  is  probably  one  of  the  most  important 
detoxicating  reactions  of  the  body  because  of  the  great  amount  of 
ammonium  compounds  that  is  constantly  being  formed  in  nitrogenous 
metabolism. 

Summary. — As  Fromm  points  out,  the  variety  of  reactions  and 
the  variety  of  defensive  substances  are  both  remarkably  small  in  num- 
ber. The  reactions  are:  oxidation  and  reduction,  h^'dration  and  de- 
hydration, and  perhaps  simple  addition  (meth^lation).  The  chief 
knov.-n  protective  substances  are  the  alkalies  of  the  blood,  proteins, 
hydrogen  sulphide,  sulphuric  acid,  glycine,  urea,  cysteine,  bile  acids, 
glycuronic  acid,  and  acetic  acid.  All  these  substances  are  normally 
present  in  the  body,  and  none  of  them  is  specific  against  any  one  poison, 
but  each  combines  with  several  poisons.  This  last  fact  is  interesting 
in  comparison  with  the  highlj^  specific  nature  of  the  immune  substances 
against  bacteria  and  their  products. 

As  far  as  we  know,  no  particular  increase  in  the  neutralizing  sub- 
stances results  from  the  administration  of  inorganic  or  organic  poisons. 
The  body  does  not  appear  to  produce  any  excessive  amounts  of  sul- 
phuric acid  in  carbolic-acid  poisoning,  or  of  glycine  when  benzoic 
acid  is  administered.  Both  substances  are  present  in  the  body  norm- 
ally, and  as  much  as  is  available  combines  with  the  poison;  if  there  is 
not  enough,  the  remaining  poison  combines  with  something  else,  or 
goes  uncombined.  In  other  words,  the  neutralizing  substances  des- 
cribed above  do  not  appear  to  be  the  result  of  any  special  adaptation 
to  meet  a  pathological  condition.  They  are  present  in  the  body  as 
a  result  of  normal  metabolism;  they  have  an  affinity  for  various 
chemical  substances,  some  of  which  happen  to  be  poisons;  if  these 
poisons  happen  to  enter  the  body,  they  may  be  combined  and  neutral- 
ized to  some  extent,  but,  as  a  rule,  very  incompletely-.     There  appears 

'^  In  this  connection  it  may  he  iiuMitioncd  tliat  the  bactericidal  power  of  the 
Mood  is  increased  if  the  blood  is  inoie  alkaline,  rlocreased  if  it  is  less  alkaline, 
than  usual. 


246  DEFENSE  AGAINST  NON-ANTIGENIC  POISONS 

to  be  no  elaborate  process  of  defense  against  the  chemically  simple 
poisons,  such  as  seems  to  be  called  into  action  by  bacterial  infection, 
and  hence  a  degree  of  resistance  or  immunity  similar  to  that  present 
after  an  attack  of  scarlet  fever  or  smallpox  does  not  exist  for  strychnine 
or  phosphorus. 

It  is  also  of  interest  to  consider  that  unicellular  organisms  may 
show  a  marked  capacity  to  increase  their  resistance  to  poisons,  as 
shown  especially  by  Ehrlich's  studies  on  trypanosomes,  which  readily 
become  immune  to  various  trypanocidal  drugs,  including  arsenic 
•compounds,  and  which  transmit  this  acquired  immunity  through  suc- 
ceeding generations.  Yeasts  and  bacteria  can  also  exhibit  increased 
tolerance  to  antiseptics,  and  Effront  found  that  yeasts  owe  their  aug- 
mented tolerance  to  fluorides  to  an  increased  content  of  calcium, 
which  precipitates  the  fluoride  which  enters  the  cells;  this  tolerance  is 
also  transmitted  to  new  generations  of  yeasts.  The  acquired  tolerance 
is  specific  in  all  these  cases,  and  may,  indeed,  be  accompanied  by 
a  decreased  resistance  to  other  poisons;  thus,  protozoa  acclimated  to 
alcohol  may  be  more  susceptible  to  other  chemicals.^'*  Paramecia 
made  immune  to  antimony  are  not  immune  to  arsenic,  and  this  specific 
immunity  is  transmitted  to  succeeding  generations  (Neuhaus).^^ 

3^  Daniel,  Jour.  Exper.  Zool.,  1909  (6),  571. 

36  Arch.  Internat.  Pharmacoydn.,  1910  (20),  393. 


CHAPTER  XI 

INFLAMMATION' 

Although  morphological  alterations  are  prominent  features  of  the 
reaction  of  the  tissues  to  local  injury  and  infection,  yet  at  the  bottom 
the  processes  of  inflammation  are  brought  about  by  and  result  in 
chemical  alterations.  The  causes  of  inflammation  are  in  nearly  all 
cases  chemically  active  substances,  but  for  the  most  part  their  nature 
is  too  little  known  to  permit  of  speculation  as  to  what  chemical  char- 
acteristic or  characteristics  a  substance  must  possess  to  exhibit  the 
power  of  causing  an  inflammatory  reaction.  Even  in  the  case  of  in- 
flammation due  to  mechanical,  thermal,  and  electrical  injuries,  it 
seems  probable  that  most  of  the  features  of  the  inflammatory  reaction 
are  brought  about  by  the  action  of  chemical  substances  produced  by 
alterations  in  the  tissue  constituents  at  the  point  of  injury,^  for  tissue 
proteins  that  have  been  altered  in  necrosis  are  chemotactic,^  as  also 
are  extracts  of  tissues. 

The  essential  features  of  inflammation,  namely,  local  hyperemia 
and  related  vascular  disturbances,  exudation  of  plasma,  migration  of 
leucocytes  and  their  phagocytic  action,  all  may  be  caused  by  the  action 
of  chemical  substances  upon  the  vessels  and  leucocytes.  Active  hy- 
peremia in  the  case  of  inflammation  is  due  to  stimulation  of  the  vaso- 
dilator nerves  or  paralysis  of  the  vaso-constrictors,  or  direct  par- 
alysis of  the  muscular  fibers  of  the  arterioles;  these  may  result  from 
mechanical,  thermal,  or  electrical  stimuli,  but  in  local  infection  the 
cause  is  usually  chemical  products  of  bacterial  growth  or  of  tissue 
disintegration.  The  escape  of  blood  plasma  (inflammatory  edema) 
appears  to  depend  upon  a  number  of  factors  (discussed  more  fully 
under  "Edema,"  Chap,  xiv)  of  which  the  most  important  seem  to  be: 
(1)  injury  to  the  capillary  walls,  produced  largely  by  the  chemical 

1  For  extensive  reviews  and  bibliography  see  Adami,  in  Allbutt's  System  of 
Medicine;  reprinted  also  as  a  naonograph,  "Inflaninuition,"  1909;  also  Opie,  Arch. 
Int.  Med.,  1910  (5),  541.  Some  interesting  ideas  are  advanced  by  Klemensiewicz, 
"Die  Entziindung,"  G.  Fischer,  Jena,  1908. 

2  Schlaepfer  (Zeit.  e.xp.  Path.,  1910  (8),  181)  finds  that  the  reduction  of  methj-- 
lene  blue  is  decreased  in  inflammatory  areas,  and  advances  the  hypothesis  that 
inflammatory  stimulants  are  o.xidation  stimulants,  inflammation  occurring  only 
when  the  amount  of  oxidation  aroused  by  the  stimulant  is  insufficient.  In  accord 
with  this  is  the  observation  of  Amberg  (Zeit.  exp.  Med.,  1913  (2),  19)  that  sub- 
stances facilitating  oxidation  reduce  inflammatory  reactions.  (See  also  WooUey, 
Jour.  Amer.  Med.  A.ssoc.,  1914  (63),  2279.)  Another  observation  of  similar  sig- 
nificance is  that  phagocytosis  is  stimulated  by  H2O2,  and  that  phagocytes  react 
to  HNC  in  the  same  wav  as  the  respiratory  center  (Hamburger,  Internat.  Zeit 
phys.-chem.  Biol.,  1915  (2),  245-264). 

3  Burger  and  Dold,  Zeit.  Immunitat.,  1914  (21),  378. 

247 


248  INFLAMMATIOX 

causes  or  products  of  the  inflammation;  (2)  increased  osmotic  pres- 
sure in  the  tissues,  due  to  increased  or  abnormal  formation  of  crystal- 
loidal  substances  with  high  osmotic  pressure  from  large  molecular 
compounds,  many  of  which  are  colloids  (proteins)  without  apprecia- 
ble osmotic  pressure;  (3)  alterations  in  the  hydration  capacity  of  the 
colloids,  whereby,  through  decrease  in  salts  or  increase  in  acidity, 
they  come  to  possess  a  greater  affinity  for  water  (M.  H.  luscher). 
By  far  the  most  characteristic  feature  of  inflammation,  however,  is 
the  behavior  of  the  leucocytes — -their  increase  in  number  in  the  blood, 
their  migration  from  the  vessels  and  accumulation  about  the  point 
of  injury,  and  their  engulfing  and  destroying  various  solid  particles, 
such  as  bacteria  and  degenerating  tissue  elements.  These  processes, 
which  seem  to  indicate  something  approaching  independent  volition 
on  the  part  of  the  leucocytes  may,  however,  be  well  explained  by  ap- 
plication of  known  laws  of  chemistry  and  physics,  without  passing 
into  the  realms  of  the  metaphysical.  This  will  be  attempted  under 
the  heading  of: 

AMEBOID  MOTION  AND  PHAGOCYTOSIS 

The  accumulation  of  leucocytes  at  a  given  point  in  the  body  indi- 
cates that  some  means  of  communication  must  exist  between  this 
point  and  the  leucocytes  in  the  circulating  blood.  No  direct  com- 
munication by  the  nervous  system  or  other  structural  method  existing, 
the  only  possible  explanation  is  that  the  communication  is  through 
the  fluids  of  the  body,  and  depends  upon  changes  in  their  chemical 
composition  or  physical  condition.  As  the  latter  generally  depends 
upon  the  former,  the  communication  is  considered  to  be  accomplished 
by  chemical  agencies,  and  called  chcmotaxis. 

Chemotaxis 

Changes  in  the  chemical  composition  of  a  fluid  have  been  shown 
frequently  to  affect  the  motion  of  living  organisms  suspended  in  it. 
One  of  the  earliest  observations  was  that  of  Engehnann,''  who  noticed 
that  Bacteriurn  ternio  suspended  in  water  tended  to  accunuilate  about 
a  l)ubble  of  oxj^gen  in  the  water.  Pfeffer^  discovered  that  the  sper- 
matozoids  of  certain  ferns  were  attracted  powerfully  by  very  dilute 
solutions  of  malic  acid,  which  is  contained  in  the  female  sperm  cell, 
inchcating  that  the  migi-ation  of  the  sperm  cells  in  the  proper  direc- 
tion depends  on  a  chemical  conuinuiication,  and  he  projiostvl  tlie  t(>rni 
('h(uuotaxis  foi"  this  phenomenon.  Strong  sohitions  of  malic  acid,  <>ii 
the  other  hand,  repelled  spermatozoids.  Cane-sugar  was  found  to  at- 
tract the  spermatozoids  of  a  certain  foliaceous  moss.  In  the  case  of 
the  mali(;  acid,  it  seems  to  be  the  anion  that  jircxhices  the  effect,  since 
salts  of  malic  acid  have  exactly  the  same  propei'ty. 

^  HotiiniHoho  ZcitiiiiK,  1S81  (39),  441. 

<- lTi)f('r,sii(!li.  iuis  (leiu  Hot.  Iiistitut  in  'riil)iii^;i'ii.  ISSI-INSS,  \U\.   1  uiul  J. 


ClIEMOTAXIS  AM)  TUOPISMS  249 

Stahl's'^  oxpcriiucnl  with  a  laij;('  jelly-like  ijjasmodiuin  (Aethal- 
ium  septicum)  growing  on  l)aik  in  wet  places,  has  become  classical. 
He  found  that  if  the  Plasmodium  was  placed  on  a  moist  surface,  and 
nearl)y  was  placetl  a  drop  of  an  infusion  of  oak  hark,  the  organism 
moved  by  tiie  process  of  protoplasmic  streaming  toward  and  into  the 
infusion.  If  a  piece  of  oak  bark  was  placed  in  the  water,  plasmodial 
arms  were  stretched  out  to  it  and  the  piece  of  bark  was  soon  com- 
pletely surrounded  by  the  organism.  These  movements  were  found 
to  occur  in  any  direction,  even  exactly  against  the  force  of  gravity. 
Other  substances,  as  acids  or  strong  solutions  of  salt  or  sugar,  wercT 
found  to  cause  the  plasmocUum  to  move  away  from  them,  although 
when  sufficiently  dilute  they  exerted  an  attraction.  A  Plasmodium 
might,  however,  move  into  a  strong  sugar  solution  if  kept  with  a 
scanty  supply  of  moisture  for  some  time,  and  after  it  had  lived  in 
such  a  strong  solution  (2  per  cent.)  for  some  time,  a  weaker  solution 
or  pure  water  was  as  injurious  as  the  concentrated  sugar  solution 
previousl}'  had  been. 

Temperature  was  also  found  to  exert  a  marked  ihermofactic  effect. 
If  a  Plasmodium  was  placed  on  a  filter-paper,  one  end  of  which  was 
in  water  at  7°,  and  the  other  in  water  at  30°,  it  would  move  toward 
the  warmer  end. 

The  Theory  of  Tropisms. — Ciliated  protozoa,  which  can  move  about  freely  in 
water,  show  very  distinct  reactions  to  stimuU  of  all  sorts.  The  first  step  in  their 
change  of  direction  of  movement  is  considered  by  many  obser\'ers  to  be  an  orien- 
tation of  the  organism  until  it  is  headed  in  the  axis  along  which  it  is  to  move. 
This  is  ascribed  by  J.  Loeb^  to  the  existence  of  a  certain  degree  of  equality  of 
irritability  of  symmetrical  parts  of  the  body.  The  stimulant,  whether  it  be  rays 
of  light,  or  diffusing  chemicals,  or  heat-waves,  moves  along  definite  lineb,  and 
the  organism  receives  at  first  unequal  stimuli  on  symmetrical  parts  of  the  body, 
unless  the  axis  of  the  organism  is  parallel  to  the  lines  of  motion  of  the  stimulant. 
As  long  as  the  stimulant  acts  on  symmetrical  parts  of  the  body  unequally,  these 
parts  will  react  unequally  until  at  length  the  body  is  swung  into  a  position  where 
the  stimulation  is  equal,  when  it  will  stay  in  this  position  and  move  along  a  line 
parallel  to  the  line  taken  by  the  stimulant.  Not  only  protozoa,  Init  much  higher 
forms,  including  some  vertebrates  are  believed  to  react  in  this  way  to  stimuli — 
e.  g.,  the  maintenance  by  fish  of  a  position  heading  up  stream.  The  above  con- 
stitutes the  so-called  ^^ theory  of  tropisms,"  and  we  have  such  reactions  to  stimuli 
of  all  sorts,  not  only  chemotropism  and  thermotropism,  but  also  heliotropism  (reac- 
tion to  light);  geotropism  (to  gravitjO,  eledropism  (to  electricityj,  thig-motropism 
(reaction  to  contact),  etc. 

The  work  done  upon  tropisms  applies  particularly  to  ciliated,  freely  motile 
organisms,  and  interests  us  less  in  connection  with  leucocytes  than  do  the  obser- 
vations on  such  forms  as  Amoeha.^  In  passing  may  be  mentioned  the  thigmotaxis 
or  thigmotropism  (reaction  to  mechanical  stimuli)  shown  by  spermatozoa,  which 
explains  their  apparently  difficult  feat  of  advancing  in  opposition  to  the  cilia  of 
the  epithelial  lining  of  the  female  generative  tract.  It  may  also  be  noted  that 
the  nature  of  reactions  of  organisms  to  various  stimuli  is  not  con.'^tant  for  even 
the  same  organism.  Copepods  (minute  Crustacea)  may  be  negatively  heliotropic 
in  the  day  and  go  away  from  the  bright  surface  of  the  water,  whereas  at  night 

«  Botanische  Zeitung,  1884  (42),  145  and  161. 
^  Comparative  Physiology  of  the  Brain.  New  York,  1900,  p.  7. 
*  For  full  details  see  Jennings  (Publication  No.  16,  Carnegie  Institute.  Wash- 
ington, 1904;  also  J.  Loeb,  "Studies  in  General  Physiology." 


250  INFLAMMATION 

the  same  animals  are  positively  heliotropic  and  swarm  to  the  surface  illuminated 
brightly  by  a  lantern.  Variations  in  heliotropism  may,  in  some  cases,  be  explained 
as  due  to  chemical  changes  that  occur  in  the  organism,  which  explanation  is  made 
more  probable  by  J.  Loeb's  experiments,  which  show  that  change  in  composition 
in  the  fluid  in  which  animals  are  suspended  may  cause  a  complete  reversal  in  their 
reaction  to  a  constant  stimulus.  Motile  bacteria  seem  to  behave  much  like  cili- 
ated protozoa  in  their  reaction  to  stimuli. 

Chemotaxis  of  Leucocytes^ 

That  leucocytes  come  to  the  site  of  an  infection  because  of  chemical 
substances  produced  by  bacteria  at  this  point,  that  is  to  say,  through 
chemotaxis,  was  first  clearly  pointed  out  by  Leber^°  in  1879,  who 
likened  the  attraction  of  such  substances  for  leucocytes  to  the  effect 
of  malic  acid  upon  spermatozoids  as  shown  by  Pfeffer.  He  found 
that  in  keratitis,  leucocytes  invaded  the  avascular  cornea  from  the  dis- 
tant vessels,  not  in  an  irregular  manner,  but  all  moved  directly  toward 
the  point  of  infection,  where  they  collected.  As  dead  cultures  of 
staphylococci  produced  a  similar,  although  less  marked,  accumulation 
of  leucocytes,  he  sought  the  chemotactic  substance  in  their  bodies,  and 
isolated  a  crystalline,  heat-resisting  substance,  phlogosin,  which  at- 
tracted leucocytes  in  animal  tissues.  He  also  observed  that  capillary 
tubes  filled  with  phlogosin  or  with  staphylococci  were  soon  invaded 
by  masses  of  leucocytes. 

Since  Leber's  experiments,  many  other  investigations  have  been 
made  showing  that  chemical  substances  of  many  different  origins  other 
than  bacterial  exert  a  chemotactic  influence  on  leucocytes.  Some  sub- 
stances are  indifferent  in  effect,  most  are  positive,  while  some  are  be- 
lieved to  repel  leucocytes;  i.  e.,  are  negatively  chemotactic. 

Negative  Chemotaxis. — Probably  the  substances  that  repel  leuco- 
cytes are  few  in  number;  Kanthack,  indeed,  doubted  the  existence  of 
really  negative  chemotactic  action  upon  leucocytes.  Verigo"  also 
considers  that  as  yet  no  actual  negative  chemotaxis  has  been  satisfac- 
torily demonstrated;  but,  by  analogy  with  the  effects  of  chemicals  on 
amebae,  ciliata,  and  plasmodial  forms,  which  all  show  a  decided  nega- 
tive chemotaxis  under  certain  influences,  it  would  seem  most  prob- 
able that  leucocytes  also  should  be  repelled  as  well  as  attracted  by 
chemicals.'^ 

N  on -bacterial  Chemotactic  Substances. — One  of  the  earliest 
significant  studies  of  the  effects  of  non-bacterial  substances  upon  chem- 
otaxis was  made  by  Massart  and  Bordet,'^  who  showed  that  products 

"  Review  of  literature  on  leucocytes  by  llelly,  Ergeb.  allg.  Pathol.,  1914(17(n),  1. 

'0  Fortschritte  der  Med.,  1888  (6),  460. 

"  Arch.  d.  M('d.  oxper.,  1901  (13),  585. 

"*  Salomonsen's  observation  (Festskrift  vcd  iiulviclscMi  af  Statrns  Scrum  Iii- 
stitut,  Kopenhagen,  1902,  Art.  XII),  that  ciliated  infusoria  wlu-ii  killed  show  a 
strong  negative  effect  on  other  ciliates,  is  of  much  interest,  particularly  as  it 
seems  to  he  tlu;  ojjposite  of  the  positively  chemotactic  effect  of  dead  upon  living 
leucocytes.  Tlu;  negative  reaction  of  different  ciliata  was  specific  for  their  own 
kind  quantitatively,  but  not  qualitatively. 

'•^  Ann.  d.  I'lnst.  Pasteur,  1891  (5),  417. 


CHEMOTAXIS  OF  LEUCOCYTES  251 

of  the  disintegration  of  leucocytes  and  otlujr  cells  had  a  strong  posi- 
tive chemotactic  influence.  They  also  corroborated  the  statement  of 
Vaillard  and  Vincent  that  lactic  acid  is  an  actively  repellant  substance, 
for  thoy  found  that  tubes  containing  a  pyocyaneus  culture,  which 
ordinarily  became  filled  with  leucocytes  rapidly,  did  not  become  in- 
vaded at  all  if  lactic  acid  was  also  added  in  a  strength  of  1 :  500,  although 
leucocytes  did  enter  when  the  dilution  was  1 :  1000. 

Gabritchevskyi^  studied  the  chemical  influence  of  a  large  number 
of  substances  on  leucocytes  and  divided  them  into  three  groups:  I. 
Substances  exerting  "negative  chemotaxis, "  including  those  that  at- 
tracted only  a  few  leucocytes. ^^  II.  Substances  with  "indifferent 
chemotaxis"  which  attracted  moderate  numbers  of  leucocytes.  III. 
Substances  with  positive  chemotaxis.  If  we  correct  the  groupings 
made  by  Gabritchevsky  we  have  the  following  classification : 

I.  Substances  negatively  chemotactic  or  indifferent: 

(a)  Concentrated  solutions  of  sodium  and  potassium  salts;  (6)  Lactic 
acid  in  all  concentrations;  (c)  quinine  (0.5  per  cent.);  {d)  alcohol  (10 
per  cent.);  (e)  chloroform  in  watery  solution;  (/)  jequirity  (2  per  cent., 
passed  through  Chamberland  filter);  {g)  glycerol  (10  per  cent,  to  1  per 
cent.);  {h)  bile;  (i)  B.  cholerae  gallinarium. 
11.  Substances  with  feeble  chemotaxis : 

(o)   Distilled  water;  (6)  dilute  solutions  of  sodium  and  potassium  salts 
(1-0.1  per  cent.);  (c)  phenol;  {d)  antipyrin;  (e)  phloridzin;  (/)  papayotin 
(in  frog);  (g)  glycogen;  {h)  peptone;  (i)  bouillon;  0)  blood  and  aqueous 
humor;  {k)  carmine. 
III.  Substances  with  strong  positive  chemotaxis: 

(a)    Papayotin   (in  rabbits);   {h)   sterilized    living  cultures  of  bacteria, 
whether  pathogenic  or  non-pathogenic. 

These  results  can  only  be  considered  as  suggestive  and  not  as  accu- 
rate findings,  in  view  of  other  contradictory  results.  Buchner^^ 
obtained  from  the  pneumohacillus  of  Friedlander,  a  protein  which  ex- 
erted a  strong  chemotactic  influence,  thus  showing  the  chemical  nature 
of  the  attraction  of  leucocytes  by  bacteria,  and  he  isolated  other 
similar  proteins  from  other  bacteria.  He  also  obtained  a  "glutin- 
casein"  from  grain  which  was  related  chemically  to  the  bacterial  pro- 
teins, and  which  was  equally  chemotactic.  The  metabolic  products 
of  bacteria,  however,  he  found  to  be  negatively  chemotactic.  Alkali 
albuminate  and  hemi-albumose  were  strongly  positive,  but  peptone 
was  not.  Glycine  and  leucine  were  found  to  be  chemotactic,  but 
urea,  ammonium  urate,  skatole,  tyrosine,  and  trimethylamine  were 
not.  It  was  also  observed  that  if  the  positively  chemotactic  sub- 
stances were  injected  subcutaneously,  they  produced  general  as  well 
as  local  leucocytosis.  The  products  of  the  action  of  serum  on  bacteria, 
"  anaphylatoxin, "  produce  inflammatory  reactions,  and  probably  are 

'^  Ann.  d.  I'Inst.  Pasteur,  1890  (4),  346. 

^*  Evidently  these  substances  were  not  all  negatively  chemotactic,  but  were 
relatively  slightly  chemotactic  or  indifferent;  yet  in  the  literature  generally  these 
experiments  have  been  cited  as  indicating  a  negative  chemotactic  influence  of  the 
substances  studied. 

'«  Berl.  klin.  Wochenschr.,  1890  (27),  1084. 


252  IXFLAMMATIOX 

important  factors  in  pathology;  the  products  of  tissue  disintegration 
have  simihir  effects.''  Certain  drugs  (notably  quinine,  morphine 
and  chloral)  when  injected  subcutaneously  seem  to  reduce  the  amount 
of  leucocj'tic  emigration  at  a  point  of  local  injury  (Ikeda).'^  In 
gas  gangrene  negative  chemotaxis  is  striking,  possibly  depending  on 
the  abundant  organic  acids  produced  by  gas  bacilli.'" 

V.  Sicherer-°  found  that  chemotaxis  of  leucocytes  may  be  observed 
outside  the  body.  If  a  tube  containing  positively  chemotactic  sub- 
stances (dead  beer-j^east  cells  and  dead  staphylococci  were  the  strong- 
est) is  placed  with  one  end  in  a  leucocyte-containing  exudate,  the 
leucocytes  pass  up  into  it  against  gravity. 

Bloch^^  demonstrated  that  carbol-gh'cerol  extracts  made  from 
each  of  the  different  viscera  and  tissues  exerted  a  positive  chemotaxis, 
discrediting  the  statements  of  Goldscheider  and  Jacob  that  only 
extracts  of  hematogenetic  tissues  showed  positive  chemotaxis.  Egg- 
albumen,  gelatine,  albumen-peptone,  and  alkali  albuminate  w'ere  also 
positive,  carbohydrates  feebly  so,  and  fat  not  at  all.  Metallic  copper, 
iron,  mercury,  and  their  salts  have  also  been  found  to  be  chemotactic 
substances,  but  it  is  very  probable  that  thej-  act  in  part  through 
destrojang  the  tissues  in  their  vicinity,  which  give  rise  to  decom- 
position-products having  a  positive  effect.  Adler,'--  however,  found 
that  bichloride  of  mercury  as  dilute  as  1 :  3000  caused  more  leucocytic 
invasion  of  a  piece  of  saturated  elder  pith  than  did  even  cultures  of 
pyogenic  bacteria. ^^ 

Aletchnikoff  observed  that  leucocytes  might,  after  a  time,  be  at- 
tracted toward  substances  that  at  first  seemed  to  repel  them.  If  the 
blood  is  full  of  toxins,  the  subcutaneous  introduction  of  bacteria  does 
not  lead  to  a  local  accumulation  of  leucocytes,  presumably  because  the 
difference  in  chemotaxis  between  the  blood  and  the  tissue  fluids  is  not 
great  enough  to  cause  emigration;  in  this  connection  should  be  men- 
tioned Pfeffer's  observation  that  B.  tcrmo  in  a  peptone  solution  will 
not  migrate  toward  another  stronger  peptone  solution,  unless  the  lat- 
ter is  at  least  five  times  as  strong  as  the  former.  Leucocytes  will 
migrate  freely  toward  substances  that  kill  them;  of  the  bacterial  prod- 
ucts the  toxins  of  pyocyaneus  and  diphtheiia  l)acilli  l)eing  especially 
destructive  and  causing  typical  karj'orriiexis.^^  Substances  soluble 
in  lipoids  are  said  by  Hamburger-'  to  increase  phagocytic  activity 
when  in  extreme  dilutions,  although  stronger  concentrations  are  highl}- 

"  See  Dold,  Arb.  Path.  Inst.  Tiihingoii,  1014  («»).  .SO. 
'».Iour.  Phamuicol.,  1916  (8),  1.37. 

'■'Sec  Kinivs-Kohcits  ;iiul  CowoU,  .lour.  Putli.  I^act..  1!)17  fJl  i.  473. 
=»  VAiwi.  f.  iiakt.,  1,S99  (2()),  3()0. 
-'  Cent.  f.  allK.  Path.,  1K9()  (7),  785. 
--  Festschr.  for  -V.  Jacolii.  1900,  New  "\  oik. 

-•H'onccrnin^  the  ctTects  of  iotlin  and  iodide.s  upon  the  ItMic.ocvtos.  see  TIeiiiz, 
Vircliow's  Aicli.,  1S99  (l.W),  44. 

2*  Schiirniann,  Cent.  f.  Pathol.,  1910  (21),  337. 

"Arch.  NY>erland.,  1912  (III,  H),  134;  Brit.  M<<.|.  ,luur..  191(1  a\  37. 


niKMOTAXIS  OF  LEUCOCYTES  253 

toxic  lor  Icucocylcs.  It  ;iii  clcclric  cunciit  is  pnsscMl  tliioii^li  two  fiii- 
goivs  there  will  be  foiiiul  more  leucocytes  in  the  tissues  of  the  catiiodc 
finger  than  in  the  anode  finger,  prosuniahly  because  the  OH-i(jns 
increase  ameboid  movement. ^^ 

Man}''  substanc(!s  have  b(>en  used  to  inciease  the  numbcjr  of  leuco- 
cytes in  the  circulating  blootl  in  the  hojjc  of  increasing  resistance  to 
infections,  a  result  that  does  not  seem  to  follow  artificial  leucocytosis 
with  any  recognizable  uniformit3\  A  compilation  of  the  literature 
on  this  subject  In-  Gehrig'-^  shows  such  contiadictory  findings  as  to 
indicate  that  most  of  the  recorded  work  is  of  little  value.  He  was 
unable  to  corroborate  the  current  statement  that  antii)yretic  drugs 
increase  the  number  of  leucocytes  in  the  blood.  Nucleinic  acid  and 
tissue  extracts  seem  to  increase  circulating  leucocytes  with  considerable 
regularity,  while  with  thorium-X  and  benzol  they  can  be  reduced  to 
almost  complete  extinction.  The  behavior  of  inflammatory  processes 
in  animals  thus  deprived  of  available  leucocytes  has  considerable 
experimental  interest. ^^^  If  less  than  1000  leucocytes  per  cubic  mm. 
are  present  in  the  blood,  no  leucocytic  exudate  can  be  produced,-^ 
although  the  other  features  of  inflammation  occur  as  usual. 

Relation  of  Cell  Types  to  Migration. — Of  the  leucocytes,  the 
most  actively  affected  by  chemotaxis  is  the  polymorphonuclear  vari- 
ety, but  not  all  substances  affect  each  variety  of  leucocyte  in  the  same 
way;  for  example,  infections  with  most  animal  parasites  result  in 
both  local  and  general  increase  in  the  eosinophilous  forms,  and  similar 
effects  have  been  obtained  by  the  injection  of  extracts  of  animal  para- 
sites. Lymphocytes  are  much  less  active,  presumabl}^  because  they 
contain  less  of  the  mobile  cytoplasm  and  consist  chiefl}^  of  the  struc- 
turally fixed  nuclear  substance.  Undoubtedly  many  of  the  cells  in 
so-called  lymphocytic  accumulations  seen  in  certain  conditions,  such 
as  tuberculosis,  are  not  lymphocytes  from  the  blood,  but  are  newly 
divided  cells  of  the  tissue.^"  The  experimental  evidence  concerning 
lymphocytic  emigration  is  very  contradictory.  Fauconnet^'  has 
found  that  tuberculin  injections  cause  in  man  general  increase  in 
leucocytes,  but  only  of  the  polymorphonuclear  form.  Long-continued 
intoxication  of  animals,  however,  may  result  in  lymphocytic  increase, 
but  local  introduction  of  the  toxin  leads  to  accumulation  of  polymor- 
phonuclear cells  and  not  lymphocj^tes.  Wolff^-  claims  that  tetanus 
and  diphtheria  toxins  produce  lymphocytosis  in  experimental  animals. 
Wlassow  and  Sepp-^^  state  that  lymphocj'tes  are  not  capable  of  ameboid 

-^  Schwyzer,  Biochem.  Zeit.,  1914  (60),  454. 

2-  Zeit.  exp.  Path.,  1915  (17),  161. 

""  See  G.  Rosenow,  Zeit.  exp.  Med.,  1914  (3i,  42. 

2'  Camp  and  Baunigartnpr,  Jour.  Exp.  Med.,  1915  (22),  174. 

30  See  resume  bv  Pappenlieim,  Folia  Hematol..  1905  {2),  815;  1906  (3j,  129. 

"  Deut.  Arch,  kliri.  Med.,  1904  (82),  1(17. 

^=  Berl.  klin.  Woi-h.,  1904  (41),  1273. 

33  Virchow's  Arch..  190t  (176),  185. 


254  INFLAMMATION 

movement  or  phagocytosis  in  the  body,  although  after  heating  to  44° 
they  may  become  motile  for  a  short  time.  Particularly  significant 
is  the  experiment  of  Reckzeh^*  who  found  that  in  lymphatic  leukemia 
with  the  lymphocytes  greatly  exceeding  the  polymorphonuclear  forms 
in  the  blood,  the  pus  from  an  acne  pustule  or  from  cantharides  bhsters 
contains  practically  no  13'mphocytes,  but  is  composed  of  the  usual 
polynuclear  forms. 

Experiments  on  the  nature  of  the  leucocytes  attracted  by  different 
chemotactic  agents  have  been  made  by  Borissow^^  and  Adler.^^  Both 
agree  in  stating  that  none  of  the  substances  tested  shows  any  special 
affinity  for  any  single  type  of  leucocytes.  Zieler^'^  observed  that  in 
the  skin  of  rabbits  exposed  to  the  Finsen  light,  active  migration  of 
lymphocytes  takes  a  prominent  part  in  the  reaction.  General  Ij^m- 
phocytosis  may  be  produced  by  certain  substances  (pilocarpine,  mus- 
carine, BaClo)  which  cause  contraction  of  the  smooth  muscles  and  force 
these  cells  out  of  the  spleen  (Harvey), ^^  but  such  a  process  has  no 
relation  to  chemotaxis.  It  is  notorious  that  infections  with  animal 
parasites  cause  both  local  and  general  increase  in  eosinophiles,  and 
we  may  even  have  local  mast-cell  leucoc3^tosis.^^ 

Tissue  cells  were  found  by  Alder  to  migrate  far  into  blocks  of  elder 
pith,  apparently  rather  later  than  the  leucocytes.  As  they  showed 
changes  of  form  indicating  ameboid  motions  he  considers  their  migra- 
tion to  be  an  active  process.  The  existence  of  the  polymorphonuclear 
forms  in  the  pith  seems  to  be  very  transient. 

The  position  taken  by  the  young  blood-vessels  and  cells  in  granula- 
tion tissue,  at  right  angles  to  the  surface,  possiblj^  also  depends  on 
chemotaxis  determining  the  direction  in  which  the  new  cells  shall  pro- 
liferate. 

Thermotaxis  of  Leucocytes. — Heat  seems  to  affect  leucocj^tes  much  as  it  does 
ameba^,  moderate  temperatures  being  positively  thermotactic.  Mendelssohn^" 
states  that  the  thermotaxis  is  most  pronounced  at  a  temperature  of  36°-39°  C. 
(97°-102°  F.),  but  is  still  marked  as  low  as  20°  C.  Temperatures  higher  than 
39°  C.  (102°  F.)  do  not  seem  to  attract  them.  Wlassow  and  Sepp-"  state  that 
motility  of  leucocytes  is  increased  by  warming  to  40°  C,  and  that  temperature 
of  42°-46°  C.  causes  the  movements  to  become  very  irregular,  with  feeble  power 
of  contraction.  Lymphocytes  are  not  motile  at  ordinary  temperature,  but  at  44° 
they  begin  to  move,  and  once  motile,  they  continue  their  motion  when  cooled  as 
low  as  35°;  this  motility  is  considered  to  be  entirely  abnormal  and  only  the  result 
of  degenerative  changes.  Murphy^-  and  his  colleagues  have  found  tliat  exjiosure 
of  animals  to  suitable  degrees  of  overheating,  leads  to  marked  lymphocj'tosis. 

»^  Zeit.  f.  klin.  Med.,  1903  (50),  51. 

"  Ziegler's  Beitrilge,  1894  (1(5),  432. 

36  PY^stschrift  f.  A.  Jacobi,  New  York,  1900. 

"  Cent.  f.  Pathol.,  1907  (18),  289. 

"Jour,  of  Physiol.,  1900  (35),  115;  see  also  Rous,  Jour.  Exper.  Med..  1908 
(10),  238. 

"See  Milchener,  Zeit.  klin.  Med.,  1899  (37),  194;  Massaglia,  Cent.  f.  Path. 
1910  (21),  534. 

"Roussky  Vratch    1903. 

<'  Virchow's  Archiv.,  1904  (176),  185. 

"Jour.  Exp.  Med.,  1919  (29),  1. 


PHAGOCYTOSIS  205 

If  mixtures  of  leucocytes  and  bacteria  sensitized  with  oj)Sonins  are  kept  at  low- 
temperature,  the  bacteria  become  attached  to  the  surface  of  the  leucocytes,  not 
being  ingested  until  the  mixture  is  wanned.^'  This  indicates  that  two  separate 
processes  are  involved  in  phagocytosis. 

Temperature  probably  plays  but  a  minor  part  in  attracting  leucocytes  in 
pathological  processes,  however.  The  local  heat  of  an  inflamed  area  is  due  chiefly 
to  the  accumulation  of  blood  in  the  part,  and  would  not  influence  the  leucocytes 
to  migrate  from  the  still  warmer  blood  into  the  ti.ssues.  Segale,^*  hciwever,  has 
demonstrated  tiiat  there  is  some  actual  heat  production  through  increased  metab- 
olism in  inflamed  tissues,  which  may  have  .some  slight  effect.  By  increasing 
motility  the  temperature  of  fever  may  favor  migration  and  phagocytosis,  and 
local  application  of  heat  to  inflamed  areas  may  induce  local  leucocytic  accumula- 
tion. In  burns  the  duration  of  the  period  of  excessive  temperature  is  usually 
too  brief  to  account  for  the  attraction  of  leucocytes  that  results;  this  accumu- 
lation is  undoubtedly  due  to  the  products  of  the  resulting  cell  degenerations. 

The  influence  of  light,  mechanical  stimulation,  and  gravity  upon 
leucocytes  seems  not  to  have  been  studied.  The  phagocytosis  of 
insoluble  non-nutritive  particles  has  been  ascribed  to  tactile  stimulation, 
but  the  details  of  the  operation  of  such  stimuli  are  unknown,  and  the 
entire  question  of  tactile  stimulation  is  unsettled.  In  experiments 
with  elder  pith  it  has  been  observed  that  leucocytes  penetrate  to  the 
center,  even  when  the  pith  contains  only  physiological  salt  solution. 
As  Adler  remarks,  it  is  difficult  to  explain  such  migration  as  due  to 
tactile  stimuli;  but  on  the  other  hand,  no  other  explanation  has  been 
offered. 

Phagocytosis  *= 

The  engulfing  of  bacteria,  cells,  tissue  products,  etc.,  by  leucocytes 
seems  to  be  but  an  extension  of  the  phenomenon  of  chemotaxis.  When 
the  substance  toward  which  the  leucocyte  is  drawn  is  small  enough, 
the  leucocyte  simply  continues  its  motion  until  it  has  flowed  entirely 
about  the  particle.  Later  the  particle  becomes,  as  a  rule,  more  or  less 
altered  within  the  cell,  unless  it  is  a  perfectly  insoluble  substance,  such 
as  a  bit  of  coal-dust.  This  action  upon  the  engulfed  object  is  un- 
doubtedly due  to  the  action  of  intracellular  enzymes. ^*^  Protozoa 
take  their  food  into  a  specialized  digesting  vacuole  which  has  been 
shown  by  Le  Dantec*^  (in  Stentor,  Paramoecium,  and  some  other  varie- 
ties) to  contain  a  strongly  acid  fluid.  Miss  Greenwood*^  has  also 
demonstrated  acid  in  several  forms  of  protozoa,  which  is  formed  under 
stimulation  of  injected  particles,  whether  nutritious  or  not.     Mouton'*^ 

^^Ledingham,  Proc.  Royal  Soc,  1908  '80),  188;  Sawtchenko,  Arch.  sci.  biol. 
1910  (15),  145. 

"  Jour.  Exp.  Med.,  1919  (29),  235. 

**  See  review  by  Metschnikoff,  Kolle  and  Wassermann's  Handb.  d.  Path.  Mik- 
roorganismen,  1913  (II),  655;  also  H.  J.  Hamburger.  "  Physikalisch-chemische 
Untersuchungen  iiber  Phagocyten,"  Bergmann,  Wiesbaden,  1912,  where  is  given 
a  full  account  of  the  author's  important  researches  on  the  principles  of  phagocytic 
behavior. 

«  See  Opie,  Jour.  Exp.  Med.,  1906  (8),  410. 

"  Ann.  d.  I'Inst.  Pasteur,  1890  (4),  776. 

"  Jour,  of  Physiol.,  1894  (16),  441. 

"  C.  R.  Acad,  des  Sciences,  1901  (133),  244. 


256  IXFLAMMATIOX 

has  been  able  to  extract  from  the  bodies  of  protozoa  (rhizopods)  a 
feebly  proteolytic  enzyme.  This  "atnibodiastase,"  as  he  calls  it,  is 
active  in  alkaline,  and  faintly  acid  media,  and  digests  colon  bacilli  that 
have  been  killed  by  heat,  but  not  living  bacilli.  This  last  fact  is 
highly  suggestive  in  connection  with  the  important  question  of  whether 
leucocytes  engulf  and  destroy  virulent  bacteria  or  only  those  that  have 
been  previously  injured  by  the  tissue  fluid.  It  was  impossible  to  se- 
cure either  invertase  or  lipase  in  extracts  of  protozoa.  Whether  bac- 
teria are  digested  in  leucocytes  by  the  same  enzymes  that  digest  the 
leucocytes  themselves  after  they  are  killed  (?'.  e.,  the  autolytic  fer- 
ments), or  by  some  specialized  enzyme  is  not  known.  Metchnikoff, 
however,  has  noted  the  localized  production  of  acid  in  the  cytoplasm 
of  leucocytes  of  the  larva  of  Triton  taeniatus.  The  eventual  excre- 
tion of  the  remains  of  the  bacteria  or  other  foreign  bodies  by  the 
phagoc3^tes  is  ascribed  by  Rhumbler  to  changes  in  the  composition  of 
the  particles  through  digestion,  so  that  they  have  a  greater  surface 
affinity  for  the  surrounding  fluids  than  for  the  protoplasm  of  the  cell. 
Calcium  and  magnesium  salts  increase  phagocytosis  and  leucocytic 
migration, ^<^  while  changes  in  osmotic  pressure  decrease  these  activi- 
ties, as  also  does  quinine  even  in  dilutions  of  0.001  per  cent.  Phago- 
cytosis cannot  take  place  in  the  absence  of  elect rol3'tes,  according  to 
Sawtchenko.^^  Fat-soluble  substances  in  general  increase  phagocyto- 
sis (Hamburger),^-  but  cholesterol  inhibits  phagocytosis.^^  (its  ef- 
fects being  suppressed  by  lecithin)  ^^  acting  apparent I3'  by  virtue  of 
its  OH  group.  Agents  facilitating  oxidation  favor  phagocj'tosis 
(Arkin).^^  Maximum  phagocytosis  occurs  at  the  normal  bodj^  tem- 
perature of  the  animal  furnishing  the  leucocytes  (Madsen  and  Wulf'  .'^ 
Phagocytosis  cannot  be  readil}-  ascribed  to  chemotaxis,  however, 
in  the  case  of  phagocj^tosis  of  perfectly'  insoluble,  chemicalh'  inert  par- 
ticles, such  as  coal-dust.  The  leucocytes  seem  to  take  up  foreign 
bodies  without  reference  to  their  nutritive  value,  absorbing  India-ink 
granules  and  bacteria  impartially  when  they  are  injected  together, 
and  loading  themselves  so  full  of  carmine  granules  that  they  cannot 
take  up  bacteria  subsequent!}-  injected.  It  is  possible  that  foreign 
bodies  first  become  coated  with  a  layer  of  altered  protein  which  then 
leads  to  phagocytosis,  but  there  is  no  sufficient  evidence  for  this  sur- 
mise.    Kite  and  Wherry^^  state  that  leucocytes  take  up  car])on  parti- 

*"  Hamburger,  Biochem.  Zeit.,  1910  (26),  GO;  Eggers,  Jour.  Infect.  Diseases, 
1909  (6),  662.  According  to  Radsma  (Arcli.  neerl.  pliysiol.,  191S  (2),  .301)  cal- 
cium salts  only  favor  phagocytosis  in  leucocytes  that  have  previously  had  their 
calcium  l)oun(l  by  citrate  or  oxalate  in  the  ])rocess  of  isolation. 

"Arch.  .sci.  Inol.  8t.  IVtershurg.  1911  (1(1),   UH  ;  1912  (17),   12S. 

"  Hamburger  and  de  llaan,  Arch.  .\na(.  und  Phv.sio!.,  19i;J,  I'hys.  .Vbt.,  p.  77 

"  Dewey  and  Nuzum,  .Jour.  Infect.  I)is.,  1914  (if)),  72. 

'■'Htuber,  liiochem.  Zeit.,  1913  (51),  211;  1914  ('>;{),  493. 

"Jour.  Infect.  Dis.,  1913  (13),  41S. 

"Over,  Dan.ske  Vid.  Selsk.  Forh.,  191t)  ((>),  339. 

"  Jour.   Infect.  Dis..  191.")  (Ki),  109. 


PHAGOCYTOSIS  257 

cles  anclTsimilar  substances  because  thc^ leucocytes  are  "sticky," 
which  presumably  is  correct,  but  what  constitutes  the  "stickiness"  and 
why  it  varies  under  the  influence  of  scrum  is  not  indicated.  Presuma- 
bly it  represents  an  altered  viscosity,  which  is  known  to  be  increased 
by  increased  acid  content  such  as  niiglit  be  produced  by  local  asphyxia.^* 
The  nature  of  mechanical  stimulation  of  cells  is  explained  by  Ostor- 
hout*"  as  a  chemical  reaction  to  rupture  of  semipermeable  cellular 
surfaces,  and  there  is  evidence  from  plant  cells  supporting  this  hy- 
pothesis, but  its  applicability  to  animal  cells  has  not  been  investigated. 
The  experiments  of  Schaeffer*^"  seem  to  show  that  amcba  exhibit 
positive  chemotaxis  towards  such  insoluble  substances  as  carbon  parti- 
cles and  glass  fragments,  even  at  a  distance,  although  the  mechanism 
is  unexplained.  Similar  investigations  have  not  been  made  with 
leucocytes. 

Not  only  leucocytes  but  tissue  cells  are  capa})le  of  moving  and  per- 
forming phagocytosis  when  properly  stimulated,  and  apparently  all  or 
nearly  all  fixed  cells  may  act  as  phagocytes  under  some  conditions. 
Their  power  of  independent  movement  is  much  less  than  their 
phagocytic  power.  Endothelial  cells  are  particularly  active  in  pha- 
gocytosis, as  also  are  the  new  mesodermal  cells  produced  in  inflamma- 
tion. Apparently  they  obey  the  same  laws  as  the  leucocytes,  and 
not  only  take  up  bacteria,  but  also  fragments  of  cells  and  tissues, 
red  corpuscles,  and  even  intact  leucocytes  and  other  cells.  Brodie''^ 
considers  that  phagocytosis  by  endothelial  cells  in  lymph-glands  is  the 
natural  end  of  the  leucocytes,  and  red  corpuscles  seem  to  have  a  similar 
fate. 

Phagocytosis  is  usually  accomphshed  solely  by  the  cytoplasm  of  the 
cells,  the  nuclei  maintaining  a  passive  role;  but,  according  to  Detre 
and  Selli,^-  the  phagocytosis  of  particles  of  lecithin  is  accomphshed  by 
the  nuclei,  which  seem  to  have  a  specific  affinity  for  this  substance. 

Giant-cell  formation  may  also  be  considered  as  the  result  of  chemo- 
taxis, the  cells  moving  toward  the  attracting  particle,  and  when  the 
particle  is  larger  than  the  cells  they  spread  out  upon  its  surface,  their 
cytoplasm  flowing  together  because  of  altered  surface  tension.  The 
peripheral  disposition  of  the  nuclei  probably  depends  on  the  fact 
that  in  ameboid  motion  the  nucleus  of  the  cell  plays  an  entirely  passive 
r61e,  being  dragged  along  by  the  cytoplasm,  and  hence  it  is  located 
most  remotely  from  the  attracting  particle.  Digestion  of  materials 
taken  into  a  giant-cell  seems  to  go  on  as  in  the  individual  cells  that 
compose  it.®^ 

^8  See  Woolley,  Jour.  Amer.  Med.  Assoc,  1914  (63),  2279. 

*'  Proc.  Natl.  Acad.  Sci.,  1916  (2),  237. 

«»  Biol.  Bull.,  1916  (31),  303. 

6'  Jour,  of  Anat.  and  Phvsiol.,  1901  (35),  142. 

6-  Berl.  klin.  Woch.,  1905  (42),  940. 

«3  See  Faber.,  Jour,  of  Path,  and  Bact.,  1893  (1),  349. 


258  INFLAMMATION 

Influence  of  the  Serum  on  Phagocytosis  (Opsonins). — Phagocytosis  of  bac- 
teria by  leucocytes  seems  not  to  be  merely  a  reaction  between  the  leucocytes  and 
the  bacteria.  Wright  and  Douglas  have  demonstrated  that  certain  substances 
in  the  blood-serum  are  necessary  to  prepare  the  bacteria  for  phagocytosis,  these 
substances  being  termed  by  them  "opsonins."  If  leucocytes  are  washed  free 
from  serum  with  salt  solution  and  let  stand  in  a  test-tube  with  such  bacteria  as 
Streptococcus  pyogenes,  Staphylococcus  pyogenes,  B.  typhosus,  B.  coli,  B.  tuberculosis, 
and  various  other  organisms,  no  phagocytosis  occurs.  If,  however,  some  serum 
from  a  normal  or  an  immunized  animal  is  added  to  the  mixture,  active  phago- 
cytosis soon  takes  place.  The  action  of  opsonins  is  also  involved  in  phagocytosis 
by  endothelium."  The  character  and  properties  of  the  opsonins  are  further 
considered  among  the  reactions  of  immunity  (Chapter  vii). 

Results  of  Phagocytosis. — After  phagocytosis  has  been  accom- 
phshed,  the  fate  of  the  engulfed  object  depends  upon  its  nature.  If 
digestible  by  the  intracellular  enzymes  it  is  soon  destroyed,  but  in 
the  case  of  engulfed  living  cells,  it  seems  probable  that  they  must  be 
first  killed — ^they  form  no  exception  to  the  rule  that  living  protoplasm 
cannot  be  digested.  This  brings  forward  the  question  of  so  much 
importance  in  the  problems  of  immunity:  Do  living  bacteria  enter 
phagocytes,  or  are  they  first  killed  by  extracellular  agencies  before 
they  can  be  taken  up?  At  the  present  time  it  seems  to  be  positively 
established  that  leucocytes  do  take  up  bacteria  which  are  still  viable, 
and  which  may  either  grow  inside  the  leucocytes  or  may  be  destroyed 
by  intracellular  processes. ^^  On  the  other  hand,  leucocytes  do  not 
take  up  extremely  virulent  bacteria,  and  hence  the  question  as  to  the 
relative  importance  played  by  the  leucocytes  and  by  the  body  fluids  is 
still  undetermined.  It  is  probable  that  phagocytosis  by  fixed  tissue- 
cells  is  of  much  less  importance  in  checking  bacterial  growth  than  is 
phagocytosis  by  leucocytes.  Thus  Ruediger's  experiments  showed 
that  emulsions  of  organs,  with  the  exception  of  bone-marrow,  do  not 
destroy  streptococci  which  are  readily  destroyed  by  leucocytes.  How- 
ever, the  phagocytic  activity  of  certain  endothelial  cells,  especially  in 
lymph  sinuses  and  the  Kupffer  cells  of  the  liver,  is  so  great  that  these 
cells  may  equal  or  surpass  the  leucocytes  in  bactericidal  power.  Leu- 
cocytes do  not  seem  to  bind  bacterial  toxins. ^^ 

Indigestible  substances  may  remain  in  cells,  particularly  in  fixed 
tissue  cells,  for  very  long  periods,  if  the  substances  are  chemically  in- 
ert. The  leucocytes  seem  to  transfer  the  indigestible  particles  which 
they  have  engulfed  to  other  tissues,  particularly  to  the  lymph-glands; 
this  is  probably  accomplished  by  phagocytosis  of  the  laden  leuco- 
cytes by  the  macrophages  of  the  lymph  sinuses,  but  how  the  insoluble 
particles  are  later  transferred  to  the  gland  stroma  or  perilymphangial 
tissues,  where  they  are  chiefly  found  in  such  conditions  as  anthracosis, 
etc.,  is  quite  unknown. 

"  Briscoe,  Jour.  Path,  and  Bact.,  1907  (12),  GO. 

"  See  Iluediger,  Jour.  Amcr.  Med.  Assoc,  1905  (44).  19S. 

"*  Pettersson,  Zcit.  Ininninitat.,  1911  (S),  498.  Koozarenko,  however,  states 
that  horse  leucocvtos  ucutrnli/.c  diphtheria  but  not  tetanus  to.\in.  (Ann.  Inst. 
Pasteur,  191.'')  (29),  190.) 


THEORIES  OF  CIIEMOTAXIS  AND  PHAGOCYTOSIS  259 

Leucocytes  contain  substances  which  are  stronfj;ly  bactericidal,  in- 
dependent of  the  action  of  the  blood  serum,  and  which  have  been 
called  endolysins;^''  they  are  resistant  to  65°  or  even  higher,  and  seem 
to  be  bound  rather  firmly  to  the  protoplasm  of  the  leucocytes,  for  they 
resist  extraction  except  by  vigorous  methods;  they  have  a  complex 
structure  like  the  amboceptor-complement  bacteriolysins  of  the  serum, 
and  are  not  specific  (Weil).^^  They  do  not  pass  through  porcelain 
filters  readil}^  are  precipitated  by  saturation  with  ammonium  sul- 
phate, and  resemble  the  enzymes  in  many  respects."^  It  is  probable 
that  the  endolysins  act  upon  bacteria  that  have  been  phagocyted,  and 
perhaps  also  upon  free  bacteria  when  liberated  in  suppuration  through 
disintegration  of  the  leucocytes.  Lymphocytes  and  macrophages  seem 
to  be  devoid  of  this  endolysin.'^" 

Phagocytosis  of  hving  virulent  bacteria  may  not  always  be  an  un- 
mixed benefit.  Besides  the  obvious  possibility  of  transporting  the 
bacteria  and  spreading  infection,  we  have  also  evidence  that  living 
bacteria  may  be  protected  through  phagocytosis,  against  the  action  of 
bactericidal  substances  in  the  blood  and  tissues  (Rous  and  Jones). '^^ 

THEORIES  OF  CHEMOTAXIS  AND  PHAGOCYTOSIS 

On  the  assumption  that  leucocytes  obej-  the  same  laws  in  their  mo- 
tions as  do  the  amebse,  studies  of  the  latter  and  of  other  forms  of 
protozoa  have  furnished  most  of  the  ideas,  hypotheses,  and  theories 
of  the  forces  involved  in  leucocytic  activities.  The  structural  rela- 
tion of  the  leucocyte  to  the  ameba  is  striking,  although  by  no  means 
complete;  the  relation  of  their  activities  is  even  closer.  Each  is  a 
microscopic,  independent,  unicellular  organism,  moving  freely  in  all 
directions  by  means  of  pseudopodia  and  protoplasmic  streaming, 
taking  other  smaller  bodies  into  its  substance  and  digesting  them, 
reacting  similarly  to  like  stimuh,  and  containing  similarly  a  nucleus 
and  many  granules.  The  differentiation  of  the  protoplasm  of  the 
ameba  into  a  clear  outer  ectosarc  and  an  inner  granular  endosarc  is 
perhaps  an  important  difference,  but  as  far  as  the  two  forms  of  cells 
have  been  studied,  the  effect  of  this  difference  in  structure  does  not 
seem  to  have  been  considered.  That  the  unicellular  protozoa,  devoid  of 
any  central  nervous  system,  and  without  any  apparent  co-ordinating 
mechanism,  seem  able  to  move  about  in  a  purposeful  way,  going  toward 
food  supplies  and  away  from  injurious  agencies,  toward  or  away  from 
hght,  heat,  and  chemicals,  has  long  attracted  the  interest  of  physi- 
ologists, particularly  as  in  these  single-celled  organisms  we  may  look 
for  the  simplest  conditions  of  existence  and  the  most  elementary  hfe 
processes.     It  seems  absurd  to  imagine  that  a  paramoeciiwi  goes  toward 

"  For  general  review  see  lOing,  Zeit.  Iramunitat.,  1910  (7).  1. 

«8Arch.  ft  Hvg.,  1911  (74),  289. 

"  Manwaring,  Jour.  Exp.  Med.,  1912  (16),  250. 

"  See  Schneider,  Arch.  f.  Hvg.,  1909  (70),  40. 

^1  Jour.  Exper.  Med.,  1916  (23),  601. 


260  INFLAMMATION 

a  dilute  acid  because  it  "likes  it,"  that  an  ameba  rejects  a  piece  of 
^lass  because  it  "does  not  taste  good,"  as  we  explain  similar  mani- 
festations in  higher  forms;  furthermore,  it  has  been  shown  by  Verworn 
"that  minute  enucleated  fragments  of  protozoan  cells  react  to  stimuli 
^ust  as  does  the  entire  cell,  and,  therefore,  it  seems  that  the  only  possi- 
ble explanation  of  movements  in  protozoa  must  be  a  direct  reaction 
of  the  stimulated  part  to  the  stimulus.  The  nature  of  the  stimulus 
and  the  nature  of  the  stimulated  substance  must  determine  the  nature 
of  the  resulting  reaction,  and  most  of  the  observations  so  far  made 
suggest  that  these  reactions  can  be  explained  according  to  the  known 
laws  of  the  physics  of  fluids.  An  ameba,  or  a  leucocyte,  may  be  looked 
upon  as  a  drop  of  a  colloidal  solution,  surrounded  by  a  delicate  sur- 
face layer  which  is  more  or  less  readilj^  permeable  to  solvents  and 
to  substances  in  solution,  and  suspended  in  a  fluid  of  quite  different 
composition. 

Siirface  Tension. — Such  a  drop  of  fluid  suspended  in  another  different  fluid 
obeys  well-known  laws  of  physics.  The  particles  of  each  fluid  are  all  under  the 
influence  of  a  very  considerable  force,  called  the  cohesion  pressure,  which  tends 
to  draw  them  together  closely.  Within  the  drop  each  particle  is  subjected  to 
this  force  alike  from  all  sides,  so  that  the  forces  neutralize  one  another,  and  each 
particle  is  as  free  as  if  there  were  no  cohesion  pressure.  But  the  particles  on  the 
surface  are  subjected  to  unequal  pressure,  for  that  of  the  fluid  outside  the  drop 
is  different  from  that  inside,  and  so  the  pressure  on  the  surface  particles  is  equal 
to  the  difference  of  the  cohesion  pressure  of  the  two  fluids;  this  constitutes  the 
surface  tension.  It  is  this  tension  that  pulls  in  upon  the  surface  continually, 
causing  it  to  tend  always  to  reduce  the  free  surface  to  a  minimum,  which  condition 
exists  perfectly  in  the  sphere.  The  amount  of  cohesion  affinity  is  very  different 
in  different  fluids,  and  therefore  some  have  a  high  surface  tension  and  some  a 
low.  When  a  substance  dissolves  in  another  the  surface  tension  is  a  resultant 
of  the  surface  tension  of  the  two  substances,  and  hence  the  surface  tension  of  a 
liquid  may  be  raised  or  lowered  by  dissolving  various  substances  in  it. 

Artificial  Imitations  of  Ameboid  Movement 

Imagine  a  drop  of  fluid  suspended  in  water — let  it  be  a  drop  of 
protoplasm,  or  oil,  or  mercury;  the  drop  owes  its  tendency  to  assume 
a  spherical  shape  to  the  surface  tension,  which  is  pulling  the  free 
surface  toward  the  center  and  acting  with  the  same  force  on  all  sides. 
The  result  is  that  the  drop  is  surrounded  by  what  amounts  to  an 
elastic,  well-stretched  membrane,  similar  to  the  condition  of  a  thin 
rubber  bag  distended  with  fluid.  If  at  any  point  in  the  surface  the 
tension  is  lessened,  while  elsewhere  it  remains  the  same,  of  necessity 
the  wall  will  bulge  at  this  point,  the  contents  will  flow  into  the  new 
space  so  offered,  and  the  rest  of  the  wall  will  contract;  hence  the  drop 
moves  toward  the  point  of  lowered  surface  tension.  Conversely,  if 
the  tension  is  increased  in  one  place,  the  wall  at  this  point  will  con- 
tract with  greater  force  than  elsewhere,  driving  the  contents  toward 
the  less  resistant  part  of  the  surface,  and  the  drop  will  move  away 
from  the  j)oint  of  increased  tension.  The  rcsemblaiice  of  these  ciianges 
of  form  and  tlic  type  of  motion  produced,  to  ameboid  movement,  is 
apparent,  and  nmch  experimenting  has  been  done  to  determine  how 


IMITATIONS  OF  AMEBOID  MOTION  Jdl 

far  tlic  processes  of  motion  as  shown  by  anuilise  and  leucocytes  can 
be  reproduced  by  fluid  drops  under  various  conditions  of  experiment, 
and  to  ascertain  if  such  ameboid  movement  of  living  cells  can  be 
entirely  explained  by  the  laws  of  surface  tension. 

Gad/-  in  1878,  pointed  out  the  resemblance  to  ameboid  motion  of 
the  changes  in  shape  observed  in  drops  of  rancid  oils  in  weak  alkaline 
solution.  These  changes  in  shape  are  due  to  the  formation  of  soaps 
which  lower  the  surface  tension  of  the  drop  in  places,  so  that  the 
fluid  flows  toward  these  places  and  produces  pseudopodium-like 
projections. 

G.  Quincke'^^  later  ascribed  the  contractions  and  other  movements 
of  amebse  to  alterations  of  the  surface  tension  of  the  living  substance 
in  relation  to  that  of  the  surrounding  medium,  believing  the  sub- 
stances responsible  for  the  alterations  to  be  albuminous  soaps. 

Biitschli^^  found  that  drops  of  "foam  structure"  made  by  mixing 
rancid  oil  and  potassium  carbonate  solution  show  "protoplasmic 
streaming"  when  placed  in  glycerol,  and  that  they  exhibit  positive 
chemotaxis  toward  soap  solution  and  other  chemicals,  the  motion  be- 
ing accompanied  by  current  formation  in  the  drops.  The  "pseudo- 
podia"  formed  by  the  drops  also  show  currents  rushing  along  their 
axes  and  returning  by  way  of  the  surface.  Heat  leads  to  increased 
activity  of  motion.  The  motions  were  ascribed  by  Biitschli  to  the 
bursting  of  some  of  the  superficial  globules  of  the  foam,  which  then 
spread  over  the  surface  of  the  drops,  lowering  its  surface  tension  at 
the  point  of  contact.  He  believed  that  ameboid  motion,  likewise, 
depended  upon  rupture  of  surface  globules  of  protoplasm,  for  the 
"foam  structure"  of  which  he  has  been  the  leading  advocate. 

Bernstein, ^^  basing  his  work  on  some  observations  of  Paalzow,  ob- 
served that  a  completely  inorganic  substance,  a  drop  of  quicksilver, 
could  be  made  to  imitate  ameboid  motion  under  the  influence  of 
chemical  changes.  If  a  crystal  of  potassium  dichromate  is  placed 
near  a  drop  of  quicksilver  in  a  nitric  acid  solution,  as  soon  as  the 
yellow  color  made  by  diffusion  of  the  dichromate  reaches  the  drop  the 
quicksilver  begins  to  show  motion  and  advances  toward  the  crystal. 
This  movement  is  due  to  local  oxidation  of  the  surface  mercury,  which 
lowers  the  tension  on  that  side  of  the  drop,  toward  which  the  mercury 
then  flows.  If  the  crystal  is  removed,  the  drop  follows,  often  flow- 
ing about  it  as  if  to  take  it  in,  but  soon  again  withdrawing  when  the 
acid  dissolves  away  the  oxide  formed  on  the  surface,  only  to  return 
again  later.  All  these  movements,  which  may  be  very  life-like,  are 
readily  explained  by  changes  in  surface  tension  that  take  place  under 
the  influence  of  the  bichromate  and  the  acid,  and  are  unquestionably 
referable  to  surface  phenomena. 

'2  DuBois  Reymond's  Arch.  f.  Physiol.,  1878,  p.  181. 
"  Wiedmann's  Annalen,  1888  (35),  580. 
"*  "Protoplasm,"  translation  bv  Minchin,  London,11894. 
'»  Pfliiger's  Arch.,  1900  (80),  628. 


262  INFLAMMATION 

Artificial  Amebae. — By  far  the  most  suggestive  experiments  on  the  simulation 
of  ameboid  activity  by  non-living  substances  are  those  of  Hhumbler  (1898)  in 
his  great  work,  "Physikalische  Analyse  von  Lebenserscheinungen  der  Zelle."'* 
On  the  assumption  that  the  living  protoplasm  was  but  a  more  or  less  tenacious 
fluid,  following  the  simple  physical  laws  of  fluids,  especially  in  relation  to  its  sur- 
face tension,  he  devised  a  number  of  experiments  to  determine  the  correctness  of 
these  views.  An  ameba  may  be  regarded  as  such  a  mass  of  viscid  fluid,  in  a 
medium  in  which  it  is  nearlj^  or  quite  insoluble;  it  is  also  constantly  undergoing 
chemical  changes  within  itself,  and  taking  substances  from  or  secreting  them  into 
the  surrounding  water.  To  reproduce  partly  these  conditions  a  drop  of  clove  oil 
is  placed  in  a  mixture  of  glycerol  and  alcohol;  the  alcohol  and  clove  oil  are  miscible, 
the  glycerol  merely  retarding  the  diffusion."^  Such  a  drop  of  oil  will  move  about, 
changing  its  form  and  sending  out  pseudopodia  much  as  an  ameba  does.  These 
movements  are  undoubtedly  due  to  changes  in  the  surface  tension  brought  about 
by  the  irregular  mixing  of  the  alcohol  and  the  clove  oil.  The  effect  of  chemotaxis 
upon  an  ameba  can  likewise  be  imitated  with  such  an  "artificial  ameba."  If 
some  stronger  alcohol  is  carefully  introduced  into  the  fluid  near  the  drop,  the 
surface  tension  on  that  side  will  be  lowered,  and  the  drop  will  flow  in  that  direc- 
tion. The  effect  of  chemical  changes  within  the  drop  upon  its  motion  may  be 
demonstrated  similarly  by  injecting  a  little  alcohol  into  the  substance  of  the  drop 
near  one  edge — the  drop  will  send  out  a  pseudopodium  on  that  side,  and  perhaps 
flow  along  in  the  direction  of  the  pseudopodium.  We  can  imagine  that  metabolic 
changes  in  the  body  of  an  ameba  may  account  for  many  of  its  seemingly  purpose- 
less movements  by  altering  surface  tension  in  some  part  of  its  circumference. 
Thermotaxis,  the  effect  of  heat  in  modifying  or  impelling  ameboid  motion,  may- 
be equally  well  demonstrated  in  such  an  "artificial  ameba,"  the  drop  being  "posi- 
tively thermotactic,"  and  flowing  rapidly  toward  a  heated  point  in  the  solution, 
because  heat  lowers  the  surface  tension. 

Even  as  highly  specialized  a  process  as  the  taking  of  food  may  be  closely  simu- 
lated experimentally.  Ameba?  seem  to  possess  the  faculty  of  selecting  substances 
that  are  suitable  for  their  food,  crawling  over  particles  of  sand,  wood,  etc.,  and 
rejecting  them  when  they  are  pushed  against  or  into  the  surface  of  the  ameba, 
which,  however,  readily  takes  up  bacteria,  diatoms,  alga^,  etc.,  digests  them,  and 
later  throws  out  the  undigested  particles.  If  there  is  any  property  of  the  ameba 
that  suggests  voluntary  action,  it  seems  to  be  exhibited  in  the  choice  of  its  food, 
although  this  is  not  so  well  developed  a  selective  process  as  might  be  expected, 
for  ameba;  will  take  up  many  harmful  objects,  and  they  may  be  made  to  fill  them- 
selves so  full  of  useless  substances  that  they  cannot  take  up  food.  However,  a 
drop  of  chloroform  in  water,  which  makes  a  good  artificial  ameba,  if  "fed"  with 
various  substances,  will  refuse  some  and  take  in  others  in  a  surprisingly  life-like 
manner.  Pieces  of  glass  or  of  wood  placed  in  contact  with  the  drop,  exert  no 
influence;  if  pushed  into  the  substance  of  the  drop,  they  carry  the  surface  ahead, 
and  on  being  released  they  are  thrown  out  with  some  force.  If  a  piece  of  shellac, 
paraffin,  styrax,  or  Canada  balsam  be  brought  in  contact  with  the  surface  of  the 
drop,  however,  the  drop  flows  around  it  immediately,  and  takes  it  within  its  sub- 
stance, where  it  is  soon  dissolved.  Even  more  strikingly  like  phagocytosis  and 
intracellular  digestion,  however,  is  the  result  of  a  similar  experiment  with  a  piece 
of  glass  covered  with  shellac;  the  chloroform  "amel)a"  takes  it  up  as  readily  as  it 
does  the  shellac  alone,  but  after  all  the  coating  is  dissolved  away  the  piece  of  glass 
is  then  cast  out  of  the  drop.  The  resemblance  to  the  engulfing,  digestion,  and 
excreting  of  indigestil)le  particles  of  bacteria,  etc.,  by  amel):r.  is  so  striking  that 
it  seems  impossible  that  there  can  be  any  fundamental  differences  in  the  two 
processes.  It  will  al.so  be  noticed  that  the  drop  takes  in  only  what  it  can  dissolve 
and  rejects  what  it  cannot. 

One  of  the  most  remarkable  actions  of  the  ameba\  which  seems  almost  cer- 
tainly the  result  of  voluntary  action,  is  this:  Oftentimes  in  feeding,  an  ameba 
gets  hold  of  a  suitable  nuiterial  which  is  in  the  form  of  a  long  tlucad,  much  too 
long  for  the  amel)a  to  surround.  It  then  proceeds  to  coil  up  tlie  (hreatl  within  its 
body,  by  stretching  a  slight  distance  along  the  thread,  bending  over,  and  forming 
a  bend  in  the  thread,  and  by  repeating  the  i)rocess  it  crowds  the  tiiread  into  a 

'»  Arch.  f.  Entwicklungsmechanik,  1898  (7),  103. 

"The  details  of  these  experiments  are  as  given  briellj'  bv  Jennings,  Jour,  of 
Applied  Microscopy,  1902  (5),  1597. 


ARTIFICIAL  AMEB/E  263 

neat  coil  within  its  body,  where  it  can  be  digested.  The  process  is  done  so  sys- 
tematically and  with  svich  evident  adoption  of  the  moans  at  hand  to  the  desired 
end,  that  it  seems  as  if  it  must  be  an  adaptation  of  tlie  amolia  to  circumstances, 
the  result  of  long  experience  or  of  heredity.  That  an  artilicial  ameba  can  per- 
form the  same  manouvers  seems  hardly  credible,  but  it  is  readily  done  with  almost 
no  dilTerence  in  detail.  If  the  chloroform  drop  is  given  a  long  fine  thread  of  shel- 
lac, it  proceeds  to  bend  the  thread  in  the  middle,  and  to  send  pseudopodia  out 
along  the  thread  to  pull  it  into  the  drop,  coiling  it  up  inside  as  the  chloroform 
softens  the  substance  of  the  thread,  until  it  is  all  contained  within  the  drop,  pro- 
vided, of  course,  that  it  is  not  too  long  (a  thread  six  times  as  long  as  the  chloroform 
drop  may  be  taken  in  completely)-  The  bending  and  coiling  of  the  thread  in 
this  experiment  is  entirely  in  accord  with  the  known  laws  and  i)henomena  of  surface 
tension. 

Fully  as  striking  an  ameboid  action  as  the  coiling  up  of  a  thread  too  long  to  be 
taken  in,  is  the  building,  by  some  of  the  protozoa  clo.sely  related  to  the  ameba 
{Difflugia)  of  a  shell  which  the  animal  seems  to  form  by  cementing  together  grains 
of  sand,  or  diatom  shells,  or  other  suitable  particles.  The  particles  are  united 
so  closely  and  fitted  together  so  well  that  they  are  almost  i)erfectly  free  from  crev- 
ices. Even  this  process  is  accurately  imitated  in  Rhumbler's  experiments.  If  a 
drop  of  oil  is  mixed  with  fine  grains  of  quartz  sand,  and  dropped  into  70  per  cent, 
alcohol,  the  grains  are  thrown  out  to  the  surface,  where  thej-  adhere  to  the  surface 
of  the  drop  and  to  one  another  exactly  as  do  the  particles  in  a  difflugia  shell.  So 
well  fitted  are  the  particles  that  the  artificial  shell  may  remain  intact  for  months, 
and  resemble  the  natural  shell  indistingui&hahly. 

Furthermore,  the  phenomenon  of  cell  division  can  be  imitated  to  some  extent 
by  oil  droplets.  Biitschli  considered  that  the  cleavage  furrow  of  dividing  cells 
represented  an  area  of  greater  surface  tension,  and  McClendon  imitated  cell 
division  as  follows:  He  suspended  a  drop  of  rancid  oil  and  chloroform  between 
water  and  salt  solution,  and  allowed  sodium  hydrate  to  flow  from  pipettes  against 
two  opposite  points  in  the  droplet,  whereon  the  surface  tension  was  lowered  and 
the  drop  bulged  at  these  points,  the  band  of  higher  surface  tension  constricting 
the  drop  between  these  two  points.  Burrows  states  that  the  changes  seen  in 
cells  dividing  beneath  the  microscope  correspond  well  to  these  experimental 
observations.'^ 

Relation  of  the  Above  Experiments  to  the  Phenomena  Exhibited  by 
Leucocytes  in  Inflammation 

The  experiments  cited  indicate  strongly,  to  saj^  the  least,  that 
amebse,  and  presumably  leucocytes,  react  to  stimuli  of  various  kinds, 
chiefly  through  the  effect  of  these  stimuli  upon  surface  tension.  The 
stimuli  may  come  from  within  the  cell,  being  in  this  case  the  result  of 
changes  in  composition  brought  about  by  metabolic  processes;  such 
chemical  products  alter  the  tension  of  the  surface  nearest  their  point 
of  origin,  causing  what  appears  to  be  spontaneous  motion.  Stimuli 
acting  from  without  may  be  chemical,  thermal,  electrical,  or  mechani- 
cal, but  in  any  event  they  act  as  stimuli  to  motion  through  their  eft'ect 
upon  surface  tension;  if  they  decrease  the  surface  tension  the  cell  goes 
toward  them;  if  they  increase  the  tension,  the  cell  moves  away.''*  The 
behavior  of  leucocytes  in  inflammation  may  be  explained  on  these 
purely  physical  grounds  very  satisfactorily,  as  follows: 

At  the  point  of  cell  injury  or  of  infection,  substances  are  produced 
that  exert  positive  chemotaxis,  as  can  be  shown  by  experiments  both 

'8  See  Trans.  Congress  Amer.  Phys.,  1913  (9),  77. 

"'  OH-ions  decrease,  H-ions  increase  the  surface  tension  of  leucocytes  (Sch- 
wyzer,  Biochem.  Zeit.,  1914  (60),  306,  447,  454),  which  may  explain  the  fact 
that  lactic  and  other  acids  exhibit  negative  chemotaxis. 


264  INFLAMMATION 

outside  and  inside  the  body;  these  substances  are  chemotactic  because 
they  influence  the  surface  tension  of  the  leucocytes,  and  since  with 
most  if  not  all  the  products  of  cell  disintegration  the  effect  is  to  lower 
surface  tension,  the  chemotactic  effect  is  positive.  As  the  chemotactic 
substances  are  produced,  they  diffuse  through  the  tissues  until  they 
reach  the  walls  of  a  capillary,  through  which  thej'-  begin  to  pass,  pre- 
sumably most  rapidly  through  the  thinnest  parts  of  the  wall,  the 
"stomata"  and  intercellular  substance.  The  leucocytes  passing  along 
in  the  bore  of  the  capillary  will  be  touched  by  the  chemotactic  sub- 
stances most  on  the  side  from  which  the  substances  diffuse;  the  sur- 
face tension  will  be  lowered  on  this  side,  causing  the  formation  of 
pseudopodia  and  motion  in  this  direction.  When  the  leucocytes  come 
in  contact  with  the  wall,  their  surfaces,  because  saturated  with  the 
chemotactic  substances,  will  have  a  tension  much  the  same  as  that 
of  the  cells  of  the  capillary  wall,  which  are  likewise  saturated  with  the 
same  substances,  and  the  two  surfaces  will  tend  to  cling  to  one  another; 
explaining  the  phenomenon  of  adhesion  of  leucocytes  to  the  capillary 
wall,  when,  according  to  the  usual  description,  "the  leucocytes  be- 
have as  if  either  they  or  the  capillary  wall  had  become  sticky."^"  Sur- 
face tension  of  the  leucocytes  will  be  least  nearest  the  points  where  the 
most  chemotactic  substances  are  entering  the  capillary,  namely,  the 
stomata;  hence  the  pseudopodia  will  form  in  this  direction  and  flow 
through  the  openings,  the  rest  of  the  cytoplasm  flowing  after  and 
dragging  the  nucleus  along  in  an  apparently  passive  manner.  Since 
it  is  the  cytoplasm  that  seems  to  be  chiefly  affected  in  these  processes, 
the  nucleus  appearing  to  be  rendered  inert  by  its  relatively  dense  and 
fixed  structure,  the  leucocytes  with  most  cytoplasm  are  most  active  in 
migration,  while  those  with  the  least,  the  lymphocytes,  are  affected 
relatively  little  or  not  at  all. 

Once  through  the  vessel  wall,  the  motion  continues  in  the  same 
manner,  toward  the  side  from  which  the  chemotactic  matter  comes, 
just  as  the  mercury  drop  flows  toward  the  crystal  of  potassium  dichro- 
mate,  or  the  drop  of  oil  flows  toward  the  alcohol.  If  the  leucocyte 
meets  a  substance  that  lowers  its  surface  tension  sufficiently,  it  will 
flow  around  the  object  and  enclose  it,  just  as  the  chloroform  drop 
flows  about  the  piece  of  shellac  or  balsam;  this  constitutes  phago- 
cytosis. The  motion  of  the  leucocyte  will  continue  in  a  forward  di- 
rection until  one  of  several  possible  things  happens :  (a)  The  leucocyte 
may  reach  a  point  where  the  chemotactic  substances  are  so  thoroughly 
diffused  that  the  effects  on  its  surface  are  the  same  on  all  sides;  there 
will  then  be  no  tendency  to  move  in  any  direction,  {b)  It  may 
r(;a(;h  a  material  that  exerts  a  marked  jiositivo  iiithience  upon  it, 
causing  much  lowering  of  the  surface  tension,  ])ul   which  is  so  large 

*"  Kr('il)i(!li  (An^h.  f.  Dcrin.atol.,  1912  (114),  SS."))  describes  as  chemical  changes 
in  the  vessel  walls  during;  the  early  stages  of  inflammation,  a  diffuse  sudanophile 
change  throughout  the  endothelial  cells,  in  the  form  of  fine,  dust-like  particles. 
Probably  this  change  dei)ends  simply  on  an  aggregation  of  the  intracellular  lipoids. 


AMEBOID  MOTION  2()r) 

that  the  cytoplasm  flowing  along  its  surface  cannot  surround  it; 
other  leucocytes  will  experience  the  same  change,  their  cytoplasm  will 
fuse  together  because  of  the  equal  lowering  of  their  surface  tension, 
and  soon  we  got  a  mass  of  leucocytes  with  fused  cytoplasm  surround- 
ing the  oliject,  forming  a  "foreign  body  giant-cell."  (c)  The  leuco- 
cyte may  reach  a  place  where  the  concentration  of  the  chemicals  is  so 
great  that  chemical  changes  are  produced  in  its  cytoplasm.  If  these 
changes  are  of  a  coagulative  nature,  the  surface  of  the  cell  will  be 
stiffened  so  that  it  cannot  migrate  further;  if  of  a  solvent  nature,  the 
leucocyte  is  destroyed,  (d)  It  may  reach  the  margin  of  an  area  where 
the  preceding  leucocytes  have  become  coagulated  or  otherwise  rendered 
immobile,  so  that  they  block  its  path,  while  it  is  held  fixed  by  the  at- 
traction on  this  side,  (c  and  d  explain  the  formation  of  solid  leu- 
cocytic  walls  about  areas  of  inflammation,  and  the  frequent  absence 
of  leucocytes  within  the  central  necrotic  areas.)  (e)  The  formation 
of  chemotactic  substances  may  cease  because  the  substance  causing  the 
inflammation  has  been  used  up,  or  because  the  bacteria  have  been 
destroj^ed,  or  from  any  of  the  causes  that  terminate  inflammation. 
Those  leucocytes  still  advancing  will  reach  a  point  where  there  is  as 
much  chemotactic  substance  behind  as  in  front — they  will  then  stop 
advancing. ^^  As  the  fluids  exuded  in  the  central  portion  continue  to 
dilute  the  chemotactic  substances  and  wash  them  out,  there  will  soon 
be  less  chemotactic  substance  in  the  center  of  the  inflamed  area  than 
there  is  farther  out,  hence  the  leucocytes  will  move  away  from  the 
center  toward  the  periphery,  following  the  chemotactic  substances 
back  into  the  blood-vessel  and  the  lymph-stream.  These  are  the 
conditions  that  exist  at  the  close  of  the  inflammatory  process,  which 
results  in  the  dispersion  of  the  leucocytes. 

General  leucocytosis  can  be  explained  equally  well  on  the  same 
grounds.  Chemotactic  substances  from  the  area  of  inflammation  enter 
the  blood-stream,  and  so,  in  a  very  dilute  form,  pass  through  the  bone- 
marrow.  The  chemotaxis  in  the  blood  will  be  greater  than  that  of 
the  marrow,  and  the  leucocytes  will  move  toward  and  into  the  blood. 
As  long  as  the  blood  contains  more  chemotactic  substances  than  the 
marrow,  leucocytosis  will  increase,  to  stop  when  the  amount  in  blood 
and  marrow  is  alike  or  when  there  is  less  in  the  blood  than  in  the 
marrow. 

Behavior  of  Tissue=cens  and  Formation  of  Giant=cells. 
The  free  cells  of  the  tissues  involved  in  inflammation  can,  of  course, 
obey  the  same  influences  as  the  leucocytes,  and  apparently  do  so  in 
so  far  as  they  are  not  checked  by  structural  impediments  to  flowing 
motion;  i  .e.,  the  more  closely  a  cell  is  related  to  a  single  drop  of  fluid 

^'  The  phagocytic  action  of  leucocytes  in  vitro  is  decreased  by  substances  that 
lower  the  surface  tension,  e.  g.  chloroform  (Hamburger,  K.  Akad.  Wetensch.,  1911 
(XIII  (2)),  892).  Ether-soluble  substances  from  bacteria  have  no  effect  on 
phagocytosis  (Miiller,  Zeit.  Immunitat.,  1908  (1),  61). 


266  INFLAMMATION 

protoplasm,  the  more  closely  does  it  resemble  in  the  simplicity  of  its 
reactions  the  "artificial  ameba."  An  illustration  of  the  chemotaxis 
of  epithelial  cells  is  furnished  by  B.  Fischer,^-  who  found  that  stained 
fats  cause  growth  and  migration  of  epithelial  cells  in  the  direction  of 
the  fat.  Cells  with  much  cytoplasm  are  best  fitted  to  move  freely,  as 
a  rule,  and  hence  we  see  chiefly  the  large  endothelial  cells  of  the  lymph 
sinuses  and  the  serous  cavities,  and  the  large  hyaline  and  granular 
cells  of  the  blood  acting  as  phagocytes,  for  phagocytosis  is  no  different 
from  ameboid  motion  which  continues  about  a  particle  until  it  is  sur- 
rounded; likewise  we  see  the  "epithelioid"  and  large  endothelial  cells 
with  their  abundant  cytoplasm  fusing  together  to  form  giant-cells. 
(Note  that  such  giant-cells  are  formed  particularly  in  conditions  in 
which  the  epithelioid  cell  is  more  abundant  than  is  the  leucocyte, 
e.  g.,  tuberculosis  and  other  chronic  inflammations.  The  cells  that 
fuse  about  an  infected  catgut  ligature  are  the  leucocytes,  for  they  are 
most  abundant  in  such  a  place.)  A  good  illustration,  also,  is  the 
giant-cell  formed  by  fusing  of  leucocytes  about  blastomyces  in  minute 
abscesses  in  the  epithelium  in  blastomycetic  dermatitis;  the  epithelial 
cells  cannot  flow  or  coalesce  well  because  of  their  abundance  of  stiff 
keratin  and  their  specialized  cell-wall,  and  hence  do  not  participate; 
the  leucocytes  are  individually  too  small  to  surround  the  fungus  cells, 
and  hence  they  flow  about  them  in  the  abscess  exactly  as  they  will  do 
experimentally  in  a  test-tube  or  in  a  guinea-pig's  abdomen  (Hektoen). 
The  method  of  growing  tissues  in  vitro  permits  of  observation  of  the 
process  of  giant-cell  formation,  and  establishes  that,  for  foreign  body 
giant-cells  at  least,  they  are  formed  by  fusion  of  wandering  cells 
(Lambert). ^^  The  formation  of  giant-cells  is,  on  this  ground,  but  an 
amplification  of  ameboid  movement  and  phagocytosis.  The  fusing  of 
the  individual  cells  is  due  to  the  lowering  of  their  surface-tension  by 
the  materials  diffusing  from  the  body  which  is  to  be  absorbed,  until 
the  surface  of  each  cell  becomes  alike,  when  the  surface  tension  at 
the  point  where  each  cell  is  in  contact  becomes  zero  and  the  cytoplasm 
runs  together. 

Objections  to  the  above  Hypothesis. — Phj'sical  explanations  of  ameboid 
movement  seem  to  fit  very  perfectly  the  known  facts  concerning  the  actions  of 
leucocytes.  There  arise  but  a  few  ditliculties  in  applying  these  laws  to  leucocytic 
action;  one  is  the  phagocytosis  of  chemically  inert  bodies,  such  as  coal  particles, 
tattooing  materials,  stone  dust,  etc.  We  know  that  anioba"  al^so  may  take  up  such 
inert  materials,  altliough  tliey  generally  refuse  them,  and  it  is  believed  that  the 
particles  exert  some  local  injury  to  the  cell  wall  that  leads  to  an  alteration  in  its 
tension.  Ameba;  seem  also  sometimes  to  excrete  a  sticky  substance  over  their 
surfaces  or  over  the  foreign  nuitter  that  is  to  be  engulfed,  which  excretion  seems  to 
be  the  result  of  surface  stimidation.  Possibly  leucocytes  do  the  same.  We  nuist 
bear  in  mind,  however,  that  the  protoplasmic  cells  luvve  much  greater  possibilities 
for  action  than  the  "artificial  ameba,"  since  within  the  jn'otoplasm  countless  cliemi- 
cal  changes  are  going  on  wliicli  nmst  cause  continual  alteration  in  surfac(>  tension; 
it  is  quite  possible  that  mere  mechanical  action  nuiy  alter  cheinica!  action  at  the 

"  Miinch.  med.  Woch.,  190G  (53),  2041. 
83  Anatomical  Record,  1912  (G),  91. 


SUPPURATION  207 

point  of  contact,  so  that  the  injurinj^  particle  niaj'  become  surrounded  throu^^h  local 
liquefaction  of  the  protoplasm. 

With  the  aineba,  unfortunately,  the  explanation  of  all  its  activities  by  purely 
physical'  analogies  is  apparently  not  so  successful.  iUthouKh  simple  pseudopodia 
may  be  produced  experimentally,  and  their  formation  explained  readil}'  on  the 
surface  tension  basis,  yet  we  find  many  forms  of  pseudopodia  in  the  great  family 
of  ameba".  Some  of  them  are  branching,  some  are  fixed  in  extension,  some  have  a 
stiff  elastic  axis.  It  would  also  l)e  difficult  to  explain  cilia  as  produced  by  changes 
in  surface  tension,  yet  we  find  in  some  protozoa  tiiat  pseudopodia  may  take  on 
the  persistence  and  action  of  cilia,  and  that  cilia  may  seem  to  change  into  pseudo- 
podia. Jennings  has  made  a  most  extended  study  of  the  relations  of  the  "Be- 
liavior  of  Lower  Organisms"*'  to  the  physical  theories  of  ameboid  motion,  and  is 
unable  to  corroborate  the  claim  that  the  processes  that  go  on  in  "artificial  ameba>" 
exactly  reproduce  tliose  of  living  ameba-,  or  to  accept  the  statement  that  living 
protoplasm  behaves  exactly  as  any  similar  drop  of  fluid  would  under  the  same 
conditions.  He  states  that  the  currents  set  up  in  artificial  ameba-  by  changes 
in  surface  tension  are  not  the  same  as  those  in  living  ameba-,  contrary  to  Rhumbler 
and  to  Biitschli.  The  movement  of  ameba,  he  maintains,  is  not  due  to  the  flowing 
of  the  contents  of  the  cell  in  a  central,  axial  current  out  into  the  pseudopodium 
and  back  on  the  sides,  as  occurs  in  the  artificial  ameba;  but  rather  to  a  rolling  for- 
ward of  the  upper  surface  over  the  anterior  edge  to  the  lower  surface,  where  it 
becomes  fixed  to  the  surface  on  which  the  ameba  is  crawling.  The  part  played  by 
surface  tension,  he  claims,  is  in  the  case  of  ameba-  a  very  sul)ordinate  one,  and  it  is 
not  sufficient  to  explain  the  movements  of  the  living  cell. 

However  the  discussion  concerning  the  amebae  may  turn,  it  must 
be  appreciated  that  there  are  some  important  differences  between  even 
the  ameba  and  the  leucocyte.  The  latter  has  by  far  the  simpler 
organization,  and  approaches  in  structure,  and  presumably,  therefore 
also  in  response  to  stimuli,  more  closely  to  the  simple  drop  of  colloid 
matter.  It  has  no  pulsating  vacuoles,  no  specialized  pseudopodia, 
never  forms  shells  or  coverings,  and  does  not  conjugate  as  do  the 
amebse.  The  external  surface  of  the  leucocyte  is  much  simpler,  an 
important  fact  in  connection  with  surface  tension  effects,  for  in  the 
leucocyte  the  surface  seems  to  be  practically  undifferentiated,  naked 
protoplasm;  whereas  in  amebae  it  is  formed  of  a  well-differentiated 
"ectosarc,"  which  has  marked  motile  powers,  being  able  to  contract 
sufficiently  to  cut  an  injured  ameba  completely  in  two.  At  the  very 
least  the  surface  tension  explanation  of  leucocytic  action  agrees  per- 
fectly with  most  of  the  observed  actions  of  leucocytes,  and  it  is  the  only 
reasonable  theory  offered.  There  seems  to  be  no  middle  ground  be- 
tween such  a  physical  theory  and  a  metaphysical  theor}^  which  would 
endow  a  single  cell,  without  organs  or  nervous  system,  with  the 
reasoning  powers  of  highly  developed  animals,  a  position  incompati- 
ble with  the  entire  evidence  of  experience. 

SUPPURATION" 

For  the  formation  of  pus  two  conditions  are  necessary:  (1)  the  ac- 
cumulation of  leucocytes,  and  (2)  necrosis  and  liquefaction  of  cells 

**  Publication  No.  16,  Carnegie  Institute,  Washington,  1904;  also  see  American 
Naturalist,  1904  (38),  G25. 

*^  Inflammatory  Exudates,  their  formation  and  composition,  are  considered  in 
Chapter  xiv. 


268  INFLAMMATION 

and  tissue  elements.  Many  leucocytes  may  be  present  in  a  tissue 
without  suppuration;  e.  g.,  erysipelas.  Necrosis  of  cells  with  their 
gradual  liquefaction  and  absorption  may  also  occur  without  suppura- 
tion; e.  g.,  infarcts,  aseptic  liquefaction  necrosis,  etc.  Hence  for  sup- 
puration to  occur  there  must  be  produced  substances  with  positive 
chemotaxis,  to  cause  accumulation  of  leucocytes,  for  if  a  necrotic  area 
is  devoid  of  leucocytes,  it  does  not  suppurate;  e.  g.,  caseous  tubercles. 
Secondly,  necrosis  must  occur,  for  digestion  and  liquefaction  of  living 
cells  and  tissues  does  not  take  place.  Only  substances  meeting  these 
requirements — -i.  e.,  causing  positive  chemotaxis  and  cell  necrosis — 
will  cause  suppuration.  Therefore,  although  bacterial  infection  is 
the  usual  cause  of  suppuration,*^  it  may  be  produced  by  many  other 
substances,  among  which  the  following  are  the  best  known:  Bacterial 
proteins,  even  from  non-pathogenic  bacteria;  oil  of  turpentine,  mer- 
cury, croton  oil,  silver  nitrate  solutions  (5  to  10  per  cent.),  and  certain 
vegetable  proteins  (vegetable  "caseins"). 

An  excellent  example  of  the  importance  of  leucocytes  for  suppura- 
tive softening  is  the  caseous  tubercle,  which  is  usually  free  from 
leucocytes  and  does  not  undergo  suppuration.  If  for  any  cause  leuco- 
cytes are  attracted  into  the  caseous  area,  softening  and  pus  formation 
promptly  occur.  Hence  Heile*'^  found  that  while  pus  from  a  "cold" 
tuberculous  abscess  will  not  digest  fibrin  and  does  not  give  the  biuret 
reaction,  both  reactions  appear  after  a  leucocytosis  has  been  brought 
about  by  injection  of  iodoform.  It  was  formerly  considered  that  the 
softening  was  due  to  the  digestive  action  of  the  enzymes  of  the  in- 
fecting bacteria,  many  of  which  were  known  to  produce  digestive 
enzymes  dissolving  protein  culture-media;  e.  g.,  Staphylococcus  pyo- 
genes. Although  to  some  extent  these  enzymes  may  be  a  factor  in 
causing  the  softening  of  the  fixed  tissues  and  of  the  killed  leucocytes, 
their  effect  is  probably  insignificant  as  compared  with  the  enzymes 
liberated  by  the  leucocytes,  as  shown  by  the  production  of  active 
experimental  suppuration  under  aseptic  conditions  with  turpentine, 
croton  oil,  etc.**  Suppuration  is,  therefore,  the  result  of  three  proc- 
esses: (1)  Necrosis  of  cells;  (2)  local  accumulation  of  leucocytes; 
(3)  digestion  of  the  necrotic  cells,  fibrin,  and  tissue  elements  by  en- 
zymes which  are  derived  from  three  sources,  as  follows:  (a)  the 
leucocytes;  (6)  the  infecting  bacteria  (if  such  arc  present);  (c)  the 
fixed  tissue-cells.  Possibly  small  quantities  of  enzymes  are  also  intro- 
duced in  the  blood  plasma,  but  these  are  probably  very  inconsiderable. 

*"  Buchner  considers  that  bacteria  will  not  produce  suppuration  unless  they 
are  broken  down  so  that  their  -pyogenic  proteins  are  released;  e.  g.,  anthrax  bacilli 
cause  suppuration  when  acting  locally,  as  in  malignant  pustule,  but  not  when 
they  are  causing  septicemia,  because  only  in  the  former  case  are  their  pyogenic 
proteins  liberated 

8^  Zeit.  klin.  Med.,  1904  (55),  508. 

**  Apparently  suppuration  may  occur  in  herpes  zoster  vesicles  in  the  absence 
of  bacteria,  according  to  the  findings  of  Kreibich  (Wien.  klin.  Woch.,  1901  (14) 
683). 


COMPOSITION  OF  PUS  269 

Normal  serum,  and  probably  also  normal  cells,  contain  antibodies  for 
the  proteolytic  enzymes  of  the  leucocytes,  and  hence  neutralization  or 
destruction  of  these  antibodies  must  be  an  important  factor  in  de- 
termining the  rate  and  amount  of  suppuration.*^ 

The  influence  of  the  antionzj^mos  is  well  siiown  by  the  rabh)it,  with 
serum  rich  in  antienzymes  and  leucocytes  poor  in  protease,  so  that 
infections  with  pus  cocci  do  not  usually  lead  to  the  formation  of  liquid 
pus  (Opie).  In  man  we  see  a  similar  relation,  in  that  exudates  rich 
in  serum  do  not  suppurate  because  the  enzymes  are  inhibited  by  the 
serum;  but  if  the  excess  of  serum  is  removed  suppuration  may  then 
occur.  With  an  excess  of  enzyme  (i.  e.,  leucocytes)  the  inhibiting 
effect  may  also  be  overcome,  and  suppuration  then  begins.  Variations 
in  the  proportion  of  leucoprotease  and  serum  antiprotease  determine, 
therefore,  the  occurrence  of  suppuration,  and  the  inflammatorj''  re- 
action is  seen  to  be  fundamentally  the  same  as  the  humoral  reactions 
of  immunity,  in  that  in  each  case  the  essential  process  is  the  provision 
of  proteolytic  enzymes  to  remove  foreign  or  abnormal  protein  sub- 
stances. In  inflammation  the  proteolytic  enzymes  are  brought  in  the 
leucocytes,  in  humoral  reactions  the  enzymes  are  present  free  in  the 
plasma.  The  antiproteases  may  be  of  the  nature  of  lipoids,  probably 
with  unsaturated  fatty  acids  ( Jobling) . 

The  proteolytic  enzymes  of  the  leucocytes  and  tissue-cells  have 
been  previously  considered  in  connection  with  the  subject  of  autolysis 
(Chap,  iii),  and  it  is  necessary  here  only  to  call  attention  to  the  fact 
that  these  enzymes  are  of  at  least  two  varieties:  (1)  Proteolytic 
enzymes  of  the  polymorphonuclear  leucocytes,  which  act  best  in  alka- 
line mechum  (Opie^");  (2)  autolytic  enzymes  of  the  tissue-cells,  which 
act  best  in  an  acid  medium  or  after  a  preliminary  acidification  (Hedin, 
et  al.).  The  mononuclear  leucocytes  contain,  like  the  tissue-cells, 
enzymes  acting  in  an  acid  medium.  The  antienzymatic  action  of 
serum  is  favored  by  an  alkaline  reaction,  but  is  altogether  lost  in  an 
acid  medium  (Opie). 

Composition  of  Pus 

Because  of  its  method  of  production,  pus  consists  of  the  follow- 
ing substances:  (1)  The  constituents  of  the  exuded  blood  plasma; 
(2)  the  constituents  of  the  leucocytes  (and  tissue-cells)  that  exist  free 
in  the  pus;  (3)  the  products  of  digestion  of  the  proteins  of  the  leuco- 
cytes and  necrosed  tissues.  All  analyses  of  pus  that  are  recorded  in 
the  literature  are  in  harmony  with  the  above  statements.  In  general 
the  analyses  consider  pus  as  composed  of  two  chief  portions,  the  pus 
corpuscles  and  the  pus  serum.  As  is  to  be  expected,  the  composition 
of  pus-corpuscles  is  simply  that  of  a  large  mass  of  leucocytes,  which 

»9See  Opie,  Jour.  Exper.  Med.,  1905  (7),  IHG;  1907  (9).  207;  Arch.  Int.  Med., 
1910  (5),  541. 

90  Jour.  Exper.  Med.,  1906  (8),  410. 


270  INFLAMMATION 

contain  minute  quantities  of  substances  taken  up  from  the  pus  serum 
by  absorption  and  phagocytosis.^^  The  old  analyses  of  pus-corpuscles 
by  Hoppe-Seyler^2  ^re  given  in  the  following  table: 

Table  I. 

Quantitative   Composition  of  Pus-cells   {in   1000    parts  of  the  dried  substance). 

I  II 

Proteins 137.621 

Nuclein 342.57  [  685.85  673.69 

Insoluble  bodies 205. 66  J 

Lecithin \  i  /i  o  oo  75 .  64 

Fat |14d.Sd  75  QQ 

Cholesterol. 74.00     72.83 

Cerebrin 51 .  99  \  i  no  ca 

Extractive  bodies 44.33/ iu^.»4 

Mineral  Substances  in  1000  Parts  of  the  Dried  Substance 

NaCl 4.35 

Ca3(P04)o 2.05 

Mg3(P04)2 1.13 

FePOi 1.06 

PO4 9.16 

Na 0.68 

K trace 

As  abnormal  constituents  of  the  leucocytes  contained  in  abscesses 
may  be  mentioned  glycogen,  fat  (from  phagocytosis  and  from  "fatty 
degeneration"  of  the  leucocytes),  and  "peptone"  (Hofmeister).^^ 

Pus  serum  differs  from  blood-serum  chiefly  in  the  substances  added 
to  it  through  the  proteolytic  changes  that  occur  in  the  pus,  and  also 
in  that  it  has  lost  its  antiproteolytic  property,  containing  instead  free 
leucoprotease.  The  fibrinogen  that  escapes  from  the  vessels  into  sup- 
purating areas  becomes  so  altered  that  pus  will  not  coagulate,  even 
upon  addition  of  fibrin  ferment  (defibrinated  blood).  The  reaction 
of  the  serum  is  usually  slightly  alkaline,  becoming  strongly  alkaline 
if  much  ammonia  is  produced,  which  occurs  especially  if  there  is  sec- 
ondary infection  with  the  organisms  of  putrefacton.  Sometimes, 
however,  lipase  derived  either  from  bacteria  or  from  the  cells  causes 
the  splitting  of  sufficient  amounts  of  fatty  acids  from  the  fats  to  make 
the  reaction  acid;  lactic  and  other  fatty  acids  are  also  sometimes 
formed.  Presumably  the  nature  of  the  infecting  organism  will 
modify  the  reaction,  for  some  (e.  g.,  staphylococcus)  cause  an  acid 
formation  in  media,  while  others  (e.  g.,  pyocyaneus)  cause  an  alkaline 
reaction.  Pneumococcus  pus  is  said  to  become  markedly  acid.^^ 
Hoppe-Seyler's  analysis  of  pus  serum  gave  the  following  results,  which 

^1  The  electrical  conductivity  of  whole  pus  is  somewhat  greater  than  that  of  blood, 
and  pus  plasma  conducts  much  more  than  whole  pus,  because  of  the  resistance  of 
the  leucocytes  (Tangl  and  Bodon,  Biochem.  Zeit.,  1917  (84),  183). 

*^  Med.-Chem.  Untersuchungen. 

"  Zeit.  physiol.  Chem.,  1880  (4),  268. 

"  Netter,  Bougault  and  Salanier,  Compt.  Rend.  Soc.  Biol.,  1917  (80),  97. 


COMPOSITION  OF  PUS  271 

show  no  considerable  deviation  from  the  composition  of  blood  plasma, 
except  in  an  increased  proportion  of  fatty  matter  and  extractive 
substances. 

Table  II 

Quantitative  composition  Plasma 

of  pus  scrum  normal 

I  II  III 

Water 913.7  905.65  908.4 

Solids 86.3  94.35  91.6 

Proteins 63.23  77.21  77.6 

Lecithin 1.50  0.561 

Fat 0 .  26  0 .  29  [  1.2 

Cholesterol 0.53  0.87  1 

Alcohol  extractives 1.52  0.73\ 

Water  extractives 11.53  6.92/  4.0 

Inorganic  salts 7 .  73  7 .  77  8.1 

Quantitatively  the  chief  abnormal  constituent  of  pus  serum  is  the 
so-called  "p^jin"  of  the  older  writers,  which  is  nucleoprotein  de- 
rived from  the  decomposing  leucocytes,  and  hence  increasing  in 
amount  progressively  with  the  age  of  the  pus;^^  it  is  characterized  by 
its  insolubility  in  acetic  acid.  The  same  substance  is  found  more 
abundantly  in  the  entire  pus,  on  account  of  the  presence  of  the  cells, 
and  when  treated  with  10  per  cent.  NaCl  solution  it  forms  a  stringy 
mass  which  was  formerly  called  "Rovida's  hyalin  substance."  Glu- 
cothionic  acid,  derived  from  the  leucocytes,  is  also  present  in  pus.^^ 
In  the  pus  serum  are  found  all  the  other  constituents  of  the. leuco- 
cytes, including  particularly  lecithin,  cholesterol,  fats  (and  soaps), 
cerebrin,  "jecorin,"  and  glycogen;  and  also  the  usual  components  of 
the  blood-serum  as  well  as  some  small  quantities  of  pigment  derived 
from  decomposed  red  corpuscles. 

The  products  of  autolysis  are  of  particular  interest,  and  they  are 
found  in  varying  amount,  but  usually  less  abundantly  than  might  be 
expected,  probably  because  of  their  solubility  and  consequent  rapid 
absorption.  Albumoses  and  peptones  seem  to  be  constantly  present 
(Shattock).^^  The  common  occurrence  of  albumosuria  during  sup- 
puration presumably  depends  on  the  absorption  of  digestion  products 
from  the  pus,^^  but  true  peptone  has  not  been  satisfactorilj-  identified 
in  the  urine.  Leucine  and  tyrosine  have  also  frequentlj^  been  found 
in  pus,^^  but  Taylor^  could  find  no  workable  traces  of  either  monoam- 

56  Strada,  Biochem.  Zeit.,  1909  (16),  193. 

5«  Mandel  and  Levene,  Biochem.  Zeit.,  1907  (4),  78. 

"  Trans.  London  Path.  Soc,  1892  (43),  225. 

°^  Literature  on  albumosuria,  see  Yarrow,  Amer.  Med.,  1903  (5),  452;  Elmer, 
ibid.,  1906  (11),  169;  Senator,  International  Climes,  1905  (IV),  series  14,  p.  85. 
See  also  "Albumosuria,"  Chap.  xxi. 

"  Miiller  (Cent.  inn.  Med.,  1907  (28),  297)  recommends  the  tyrosine  reaction 
with  MiUon's  reagent  as  a  means  of  dififerentiating  tuberculous  from  ordinary 
pus,  the  former  not  giving  the  reaction  because  of  lack  of  leucocytic  enzymes; 
but  there  is  disagreement  as  to  the  constancy  of  this  reaction  in  pus  (Dold,  Deut. 
mod.  Woch.,  1908  (34),  869). 

1  Univ.  of  California  Publications  (Pathol.),  1904  (1),  46. 


272  INFLAMMATION 

ino-  or  polyamino-acids  in  a  liter  of  pus,  which  may  depend  on  their 
having  been  either  absorbed  or  transformed  into  ammonium  com- 
pounds. Presumably  this  is  in  part  the  explanation  of  the  large 
urea  excretion  in  persons  with  extensive  suppuration,  as  observed  by 
Ameuille.-  From  the  nucleoproteins  'purine  bodies  are  formed  and 
may  be  found  in  the  pus.  The  relation  of  the  purine  bases  to  local 
leucocytosis  is  shown  by  Heile,^^  who  found  in  cold  tuberculous  ab- 
scesses a  proportion  of  purine  bases  equal  to  0.5  per  cent.,  in  similar  ab- 
scesses after  injection  of  iodoform,  1.57,  and  in  acute  suppuration, 
10.7.  Spermin  crystals  are  also  occasionally  found  in  old  pus  col- 
lections.^ Free  fatty  acids  and  volatile  fatty  acids,  such  as  butyric, 
lactic,^  valerianic,  and  formic,  have  been  found.  Products  of  bac- 
terial activity,  such  as  bacterial  proteins  and  pigments  (e.  g.,  pyo- 
cyanin),  may  also  be  present.  It  is  probable  that  in  many  instances 
these  autolytic  products  are  bactericidal,  and  thus  help  to  terminate 
the  infection.  Direct  tests  have  shown  that  the  autolysate  of  fibrin  is 
bactericidal  for  staphylococci  and  streptococci.^  See  also  discussion 
of  "Autolysis  of  Exudates"  (Chap.  iii). 

All  the  numerous  enzymes  of  the  blood  plasma,  the  leucocytes  and 
the  tissue-cells,  are  present  in  pus.  Thus  Achalme®  found  evidence 
of  the  presence  of  the  following  enzymes  in  pus:  proteolytic  en- 
zymes,^ lipase  (splitting  monobutyrin),  diastase,  rennin  (coagulating 
milk),  gelatinase,  catalase,  and  oxidase,  the  last  being  very  abundant. 
These  seem  to  exist  chiefly  in  the  leucocytes,  the  pus  serum  being 
quite  free  from  them.  No  evidence  could  be  found  of  enzymes  act- 
ing on  amygdalin,  saccharose,  inulin,  or  lactose.  Fibrin  ferment  is 
said  to  be  absent  from  pus,  which  is  quite  surprising  in  view  of  the 
fact  that  this  enzyme  is  generally  considered  as  being  derived  chiefly 
from  the  leucocytes.  Presumably  the  bacteriolytic  "endolysins"  of 
the  leucocytes  are  also  present  in  pus. 

There  is  little  difference  in  the  effect  on  metabolism  produced  by  a 
sterile  suppuration  and  one  due  to  localized  bacterial  infection  (Cooke 
and  Whipple),^  one  of  the  chemical  features  in  each  being  a  precipitous 
and  sustained  rise  in  the  urinary  N  excretion.  Presumably  the  reac- 
tion in  both  cases  results  from  toxic  products  of  protein  cleavage, 
rather  than  from  bacterial  secretions  in  the  case  of  septic  inflanmiation. 
Probably  only  part  of  the  excessive  urinary  N  comes  from  the  local 
injury,  the  greater  part  being  derived  from  toxicogenic  destruction  of 
tissue  proteins. 

i^Bull.  Acad.  M(:^d.  Paris,  1917,  (78),  8. 

3  See  Williams,  Boston  Med.  and  Surg.  Jour.,  1901  (145),  355. 

''  d-lactic  acid  i.s  a  constant  constituont  of  i)us  from  the  pleura  (Ito,  Jour. 
Biol.  Chom.,  1910  (2(5)    173). 

"  Bilancioni,  Arch,  (li  Farmacol.,  1911  (11),  491. 

8  Compt.  Rend.  Soc.  Biol.,  1899  (51),  568. 

^  Concerning  proteolytic  enzymes  of  pus  see  Opie,  Jour.  Exper.  Med.,  1906  (8), 
410. 

8  Jour.  Exp.  Med.,  1918,  (28),  222. 


COMPOSITION  OF  SPUTUM  273 


Sputum" 


The  chomistiy  of  sputum  may  be  properly  considered  in  this  con- 
nection. In  reaction,  sputum  is  ordinarily  alkaline,  but  in  case  of 
marked  bacterial  decomposition  in  cavities  the  reaction  may  become 
acid.  Its  specific  gravity  varies  from  1.008  to  1.026,  usually  varying 
directly  with  the  number  of  leucocytes;  the  average  specific  gravity 
is  about  1.013  .  The  greenish  color  frequently  observed  depends  gen- 
erally upon  blood-pigment  (except  in  case  of  icterus),  although  in 
some  instances  the  pigment  is  of  bacterial  origin.  Renk'°  has  studied 
the  proteins  of  sputum  with  special  reference  to  the  loss  of  protein  to 
the  body  and  its  relation  to  cachexia.  In  three  patients  (consump- 
tives) studied,  the  daily  amount  of  sputum  of  two  averaged  145 
grams  for  each;  for  the  third  it  was  82  grams.  This  contained  (aver- 
age) 5  to  6  per  cent,  of  soHds;  including  mucin,  2-3  per  cent.;  protein, 
0.1-0.5  per  cent.;  fat,  0.3-0.5  per  cent.;  ash,  0.8-0.9  percent.  The 
daily  loss  of  nitrogen  was  0.75  gram,  which  eciuals  about  6  per  cent,  of 
the  total  daily  nitrogen  output  of  persons  under  condition  of  starva- 
tion." Wanner^-  found  characteristic  variations  in  the  amount  of 
protein  in  sputum  from  different  conditions,  as  follows:  in  bronchitis 
the  amount  of  protein  is  very  small ;  in  bronchiectasis  protein  is  pres- 
ent, but  the  amount  of  uncoagulable  nitrogen  (due  to  autolysis)  is 
relatively  large;  in  phthisis  as  well  as  in  bronchiectasis  the  amount  of 
protein  does  not  exceed  1  per  cent.;  in  pneumonia  it  may  reach  3  per 
cent.,  but  it  is  highest  in  pulmonary  gangrene.  Any  protein  content 
that  causes  more  than  a  shght  turbidity  on  boihng  indicates  an 
inflammation;  e.  g.,  in  case  of  doubt  between  a  chagnosis  of  pneumonia 
and  infarct  a  high  protein  content  speaks  for  the  former.  Rogers^^ 
stated  that  the  sputum  in  every  case  of  tuberculosis  shows  albumin,'^ 
but  this  has  been  questioned,  especially  as  to  chronic  or  quiescent 
cases. ^*  Albumin,  or  better,  coagulable  protein  is  also  present  in  the 
sputum  of  patients  with  pulmonary  edema  and  pleurisy.  According 
to  Works^^  in  active  tuberculosis  there  is  usually  0.2  per  cent,  or  more 
of  coagulable  protein  in  the  sputum.     The  mucin  of  sputum  yields 

9  Complete  bibliography  given  by  Ott,  "Chem.  Pathol,  der  Tuberc,"  Berlin, 
1903;  Falk,  Ergebnisse  Physiol.,  1910  (9),  406;  Plesch,  Hanb.  d.  Biochem.,  1908 
(HI  (1),  7. 

'"Zeit.  f.  Biol.,  1875  (11),  102. 

11  Plesch  (Zeit.  exp.  Path.  u.  Ther.,  1906,  Bd.  iii,  July)  found  that  4.8  per 
cent,  of  all  the  absorbed  calories  were  lost  in  the  sputum  in  an  advanced  case  of 
phthisis.  Under  similar  conditions  the  amount  of  salts  excreted  by  the  sputum 
may  equal  or  exceed  that  in  the  urine  (Falk,  loc.  cit.).^ 

12  Deut.  Arch.  klin.  Med.,  1903  (75),  347. 

"  Presse  Med.,  1910  (18),  289;  1911  (19),  409;  also  Ganz  and  Hertz,  iUd., 
1911  (19),  41;  Kaufmann,  Beitr.  lOin.  d.  Tuberk,  1913  (26),  269;  Hempel-Jorgensen, 
ibid.,  p.  392. 

1^  Review  by  Cocke,  Amer.  Jour.  Med.  Sci.,  1914  (148),  724. 

15  Fischberg  and  Felberbaum,  Medical  Record,  Oct.  21,  1911;  Acs-Nagy,  Wien. 
klin.  Woch.,  1912  (25),  1904. 

1'  Jour.  Amer.  Med.  Assoc,  1912  (59),  1537. 

18 


274 


INFLAMMATION 


33.6  per  cent,  of  glucosamin  when  split  with  HCl,  which  gives  an  in- 
dex of  the  quantity  of  mucin;  this  is  highest  in  chronic  bronchitis  and 
lowest  in  pneumonia  and  phthisis.  Kossel  found  0.1-0.33  gm.  of 
nucleins  in  the  sputum  dailJ^ 

The  following  table  by  Bokay  (taken  from  Ott)  gives  the  propor- 
tion of  the  organic  constituents  of  sputum  in  parts  per  thousand: 

Table  III 


Bronchitis 

in 

typhoid 


Fibroid 
phthisis 


Phthisis, 

early  in 

apex 


Phthisis,   [    Phthisis,       Phthisis, 
cavities     \  advanced  !  advanced 


Fatty  acids  as  fat 
Free  fatty  acids . . 

Soaps 

Cholesterol 

Lecithin 

Nuclein 

Protein  


0.224 

trace 

traces 

traces 

traces 

traces 

0.898 


0.845 

0.184 

0.380 

0.4 

traces 

0.102 

2.040 


0.462 
0.521 
0.430 
1.617 
1.543 


2.468        3.468 


0.370 
0.537 
0.172 


4.430 


0.307 
0.516 
1.160 
1.165 
0.260 
3.455 


9.725 
0.902 
3.973 
0.141 
1.245 
0.489 
5.115 


On  account  of  the  digestion  of  the  exudates  by  the  leucocj'tes, 
sputum  contains  proteoses,  peptones,  and  amino-acids,  generally  in 
proportion  to  the  richness  of  the  exudate  in  leucocytes;  they  are, 
therefore,  most  abundant  in  pneumonia.  Simon^^  found  considerable 
albumose  in  phthisical  sputum,  but  no  nucleohiston  or  free  histon. 
In  febrile  cases  of  tuberculosis  the  amount  of  albumose  may  exceed 
the  coagulable  albumen,  which  rarely  exceeds  one  per  cent,  of  the 
moist  weight. ^^  Staffregen,  however,  could  find  no  true  peptone  in 
phthisical  sputum,  but  Stadelmann^^  found  that  such  sputum  con- 
tained enzymes  hydrolyzing  fibrin,  and  attributed  this  largely  to 
bacteria.  Probably  most  of  the  enzymes  present  in  sputum  come 
from  the  leucocytes.  In  the  early  stage  of  pneumonia  the  sputum 
has  no  proteolytic  action,  presumably  because  inhibited  by  the  large 
amount  of  serum  present;  but  with  resolution  active  proteolytic  prop- 
erties appear  (Bittorf).^''  In  tuberculosis  sputum  the  tryptic  and 
antitryptic  properties  fluctuate,  and  lipase  is  absent  (Eiselt).-^  Pneu- 
monic sputum  before  the  crisis  has  but  slight  action  on  peptids,  but 
acquires  marked  peptolytic  activity  thereafter.-'-  Most  sputa  con- 
tain enzymes  spHtting  casein  and  polypeptids.-^  Sputum  may 
contain  indole,  derived  either  from  the  putrefying  proteins  or  excreted 
from  the  blood. ^^ 

1'  Arch.  exp.  Path.  u.  Pharm.,  1903  (49),  449. 

'«  Prorok,  Miinch.  med.  Woch.,  1909  (56),  2053. 

i»  Zeit.  klin.  Med.,  1889  (10).  128. 

"  Deut.  Arch.  klin.  Med.,  1907  (91),  212. 

2' Zeit.  klin.  Med.,  1912  (75),  91. 

"  Abderhalden,  Zeit.  phvsiol.  Chem.,  1912  (78),  344. 

"  Maliwa,  Deut.  Arch.  klin.  Med.,  1914  (115),  407. 

"  Binda  and  Cassarini,  Gaz.  Med.  Ital.,  1913  (64),  461. 


I 


COMPUSITIOX  OF  SPUTUM  275 

Tho  amount  of  fats  seems  to  (icpoiul  directly  ui)on  the  number  of 
pus-corpuscles  and  the  age  of  the  pus  (i.  e.,  the  amount  of  fatty  de- 
generation). Jacobson  found  from  0.08  to  l.G  grams  of  fatty  matter 
per  day,  containing  on  an  average  14.70  per  cent,  of  soaps,  15.79  per 
cent,  of  higher  fatty  acids,  0-10  per  cent,  of  water-soluble  fatty  acids, 
13.58  per  cent,  lecithin,  and  10.49  per  cent,  cholesterol. 

As  to  the  inorganic  substances,  Bamberger  found  two  types  of  spu- 
tum, catarrhal  and  inflammatory.  In  the  inflammatory  there  is  a 
deficiency  in  alkali  phosphate,  SO3  constitutes  more  than  8  per  cent. 

of  the  salts,  and  the  ratio, -^Y)  equals  ..•     In    catarrhal    sputum 

the  alkali  phosphates  constitute  10-14  per  cent.,   j^  ^   =  ^q'  ^"d  the 

SO3  is  from  0.6-1.2  per  cent.  Chlorine  is  about  the  same  in  both 
forms.  These  differences  are,  however,  not  as  constant  as  Bamberger 
believes,  according  to  several  later  investigations.  The  results  of  his 
analyses  are  shown  in  the  following  table: 

Table  IV 


Chronic  Acute 

phthisis  phthisis 


Water I       94.55       |       93.38 

Organic  matter 4 .  67  6 .  88 

Inorganic  salts 0 .  78  0 .  74 


One  hundred  parts  of  the  salts  contain: 

Chlorine 35.78  33.40 

SO.,  0.70  0.80 

P2O6 13.05  14.15 

K2O  24.07  19.99 

NaoO 27.90  31.69^ 

Calcium  phosphate 1-63  4 .  32'* 

Iron  phosphate 0.09  0.14 

Magnesium  phosphate 1 .  20  .... 

Ca  and  Mg  carbonate  and  sulphate 1 .  74  0 .  22 

Silicic  acid 0.9  0.3 

-^  Including  magnesium. 


CHAPTER  XII 

THE   CHEMISTRY   OF   GROWTH  AND   REPAIR 
PROLIFERATION  AND  REGENERATION 

The  factors  that  incite  cells  to  proliferate,  as  well  as  those  that 
cause  the  cessation  of  proliferation  after  it  has  once  started,  are  too 
entirely  unknown  to  permit  of  speculation  as  to  their  exact  nature. 
It  seems  probable,  however,  that  they  are,  as  Ziegler  says,  "identical 
with  the  stimuli  which  excite  or  increase  functional  and  nutritive 
activity,"  and  these  are  certainlj'  in  manj''  instances  of  chemical  na- 
ture. Thus  the  application  of  various  irritating  substances  in  not 
too  concentrated  a  form  (e.  g.,  painting  the  skin  with  iodin)  may 
lead  to  proliferation  without  causing  discernible  degeneration  of  the 
cells.  Mallory's^  observations  on  the  phenomena  of  proliferation 
and  phagocytosis  show  that  the  same  bacterial  products  which  destroj' 
the  cells  when  concentrated,  when  sufficiently  dilute  cause  prolifera- 
tion of  similar  cells.  Carnot  and  Lavlievre^  have  obtained  evidence 
that  actively  growing  kidney  tissue,  whether  fetal  or  adult  regener- 
ating kidney,  contains  something  which  is  capable  of  stimulating 
growth  of  renal  epithelium  when  injected  into  other  animals.  Numer- 
ous dyes  are  known  to  stimulate  cell  growth  greatly,  {e.  g.,  the  growth 
of  epithelium  into  oils  containing  sudan  III,  etc.)  and  sometimes  seem 
to  lead  by  virtue  of  this  fact  to  cancer  growth  (e.  g.,  cancer  of  the  bladder 
in  dye  workers).  Chemical  products  from  decomposition  of  vegetable 
matter  have  a  particularly  active  stimulating  effect,  so  that  what 
seem  to  be  true  cancers  have  been  experimentally  produced  by  paint- 
ing the  ears  of  rabbits  with  tar  (Yamagiwa).  Manj^  other  instances 
of  proliferation  in  response  to  chemical  stimuli  might  be  cited, 
but  in  nearly  all  cases  it  is  extremely  difficult  to  determine  that 
the  proliferation  is  not,  after  all,  reparative  in  compensation  for 
degenerative  changes,  and,  therefore,  possibly  obeying  some  other 
biological  law  than  that  of  a  simple  reaction  to  a  chemical  stimulus. 

Perhaps  the  most  striking  example  we  have  of  growth  stimula- 
tion by  chemical  agencies  is  furnished  by  the  proliferation  and  hyper- 
trophy which  take  place  in  the  uterus^  and  mammary  gland  during 
pregnane}'.  The  sanu;  phenomena  can  be  produced  by  injecting  the 
lipoid  fraction  of  extract  of  placenta  and  corpus  lutcum  (Frank).* 

1  Jour.  Exp.  Med.,  1900  (5),  15. 
-'  Arch.  M('d.  E.xper.,  1907  (19),  388. 

■'See  Leo  Loeb,  Jour.  Amer.  Med.  Assoc,  1908  (50),  1897;  1915  (04),  726. 
*  Jour.  Anier.  Med.  Assoc.,  1920  (74),  47. 

27() 


I'h'OLIFl'Jh'ATlOX  AM)  UEGEN ERATION  '111 

Even  transplanted  bits  of  uterine  tissue  are  stimulated  to  j^row  Ijy 
these  substances,  thus  excluding  possible  nervous  control  of  growth.* 
Dried  placenta  fed  to  mothers  also  increases  the  rate  of  growth  of  the 
suckling  infant  (Hammett).*^  The  nature  of  the  growth-stimulating 
agency  in  placenta  and  corpus  luteum  is  unknown  but  it  strongly 
resists  chemical  agents.  However,  it  may  be  pointed  out  that  tethelin, 
described  by  Robertson^  as  the  growth-promoting  substance  of  the 
hypophysis,  is  soluble  in  lipoid  solvents.  Acromegaly  and  gigantism, 
give  evidence  that  even  far  more  than  normal  growth  ma}'  be  produced, 
presumably  through  the  agency  of  an  internal  secretion  of  the  hypo- 
phesis,  but  whether  tethelin  actually  is  the  substance  responsible  is 
at  present  unknown.  This  substance  is  obtained  from  the  anterior 
lobe,  about  10  mg.  for  each  gland,  and  contains  1.4  per  cent,  of  phos- 
phorous. It  is  said  to  retard  growth  of  animals  before  adolescence 
and  to  increase  post-adolescent  growth;  also  it  has  been  reported  that 
wound  repair  is  stimulated  by  tethelin.*  At  this  time,  however,  the 
status  of  tethelin  is  not  fully  determined.  The  influence  of  the  other 
ductless  glands  on  growth  is    discussed    further   in    Chapter    xxii. 

The  studies  by  Whipple  and  his  colleagues  on  the  repair  of  the  liver 
after  extensive  chloroform  necrosis  indicate  that  a  mixed  diet  rich  in 
carbohj'drate  is  more  effective  in  facilitating  this  repair  than  meat  or 
fat,  and  that  thyroid  extract  does  not  stimulate  repair.^  Also  the 
healing  of  wounds  is  more  rapid  in  meat-fed  than  in  fat-fed  dogs. 
(Clark)  ^^  Attempts  to  find  specific  substances  that  will  cause  in- 
creased rate  of  wound  healing  have  so  far  been  unsuccessful."  Re- 
generation of  blood  protein  after  hemorrhage  is  said  to  be  most  rapid 
on  a  protein  rich  diet.^-  When  an  incomplete  protein,  ghadin,  is  the 
sole  protein  of  the  diet  the  hemoglobin  is  not  regenerated. 

Although  proper  nutrition  is  necessary  for  cell  proliferation,  yet  it 
does  not  seem  that  excessive  nourishment  can  lead  to  excessive  cell 
multiplication,  or  by  itself  cause  cell  proliferation  to  take  place. 

Oxygen  and  certain  inorganic  salts  are  essential  for  cell  division 
even  in  the  lowest  forms,  and  among  such  simple  organisms  as  sea- 
urchins  and  certain  other  marine  forms  segmentation  of  the  unfertilized 
ova  may  be  incited  bj^  changes  in  osmotic  concentration,  leading 
eventually  to  formation  of  perfect  larvae  (J.  Loeb,  et.  al.).''-^  In  lower 
animals  very  dilute  solutions  of  alkalies  stimulate  the  rate  of  cell 
growth  and  somewhat  higher  concentrations  cause  extremely  irregular 
cell  division;  in  mammals  the  feeding  of  alkalies  causes  great  wasting 

"'  Frank,  Surg.  Gyn.  Obst.,  1917  (25),  329. 

"  Jour.  Biol.  Chem.,  1918  (36),  569;  Endocrinology,  1919  (3),  307. 

'  See  Jour.  Exp.  Med.,  1916  (23),  631. 

^  Review  by  Barney,  Jour.  Lab.  Clin.  Med.,  1918  (3),  480. 

=>  See  Arch.  Int.  Med.,  1919  (23),  689. 

'"  Bull.  Johns  Hopkins  Hosp.,  1919  (30),  117. 

'iSee  DuNoiiy,  Anaer.  Jour.  Physiol.,  1919  (49),  121. 

'2  Kerr  et  al,  Amer.  Jour.  Physiol.,  1918  (47),  456. 

'^See  J.  Loeb,  Studies  in  General  Physiology,  Chicago,  1905. 


278  THE  CHEMISTRY  OF  GROWTH  AND  REPAIR 

as  if  through  cell  stimulation.'^  The  products  of  nuclein  hydrolysis 
are  said  to  stimulate  cell  growth. ^^  Potassium  salts  seem  to  be 
particularly  important  for  proliferating  cells,  and  Beebe  and  also 
Clowes  and  Frisbie^®  have  found  that  actively  growing  malignant 
tumors  are  rich  in  potassium  and  poor  in  calcium,  whereas  in  slow- 
growing  tumors  the  reverse  is  the  case.  Dennstedt  and  Rumpf^'^ 
also  found  that  in  hypertrophy  of  the  heart  the  amount  of  potassium 
is  increased,  while  in  chronic  degeneration  of  the  myocardium  the 
calcium  and  magnesium  are  usually  increased.  The  proportion  of 
nitrogen  in  the  different  parts  of  the  heart  is  not  changed  during 
hypertrophy  (Bence),^^  but  the  amount  of  NaCl  is  much  increased 
in  hypertrophy.^^ 

Chemical  studies  of  proliferation  are  lacking, ^^  except  in  regard  to 
the  development  of  the  embryo,  etc.^^  New  tissues  differ  from  adult 
tissues  in  having  a  large  proportion  of  water,  and  in  having  a  larger 
proportion  of  the  ''primary"  cell  constituents  and  a  smaller  propor- 
tion of  the  various  secondary  constituents,  since  these  last  are  largely 
products  of  the  activity  of  the  adult  cell.  Of  the  primary  constitu- 
ents, the  proportion  of  the  nucleoproteins  is  particularly  high,  and  a 
number  of  interesting  facts  concerning  the  nucleoproteins  in  cell  di- 
vision have  been  determined.  Most  important,  perhaps,  is  the  clas- 
sical observation  of  Miescher,  who  found  that  during  the  migration 
of  salmon  up  stream  to  the  spawning  grounds,  during  which  time  no 
food  is  taken,  the  proteins  of  the  muscular  tissue  become  largely 
transformed  into  the  protamin  type  of  protein  (characterized  by  con- 
taining large  proportions  of  the  polyamino-acids,  such  as  argininc 
histidine,  and  lysine),^-  which  unite  with  nucleic  acids  to  form  the 
abundant  nucleoprotein  of  the  spermatozoa  and  ova."^  Whether  such 
a  transformation  of  proteins  occurs  in  mammalian  cells  during  cell 
multiplication  cannot  be  stated,  but  certainly  from  some  source  an 
additional  supply  of  nucleoprotein  is  derived.     Developing  sea  urchin 

1^  Moore  et  al,  Biochem.  Jour.,  1906  (1),  294;  1912  (6;,  162. 

'*  Calkins  et  al,  Jour.  Infect.  Dis.,  1912  (10),  421. 

1^  See  "Tumors,"  Chap.  xix. 

1^  Zeit.  klin.  Med.,  1905  (58j,  84. 

18  Zeit.  klin.  Med.,  1908  (66),  441. 

'9  Rzentkowski,  ibid.,  1910  (70),  337. 

"<*  The  composition  of  qranulalion  tissue  has  been  determined  by  Hirsch  (Amer. 
Jour.  Med.  Sci.,  1920  (159),  356,  who  analyzed  the  "castration  granulomas"  of 
swine.  These  are  large  inflammatory  tumors,  probably  resulting  from  subacute 
infection  of  the  operation  wound,  and  consist  of  dense  fibrous  tissue  with  fow 
cells  and  a  scanty  blood  supi)ly,  but  sometimes  more  or  less  edematous.  His 
figures  are  as  follows:  Water  81.9  per  cent.;  solids,  18.1;  lipins,  2.3;  proteins,  14.4. 
The  protein  contained  sulphur,  0.34  per  cent;  phosphorus,  0.32,  purine  N,  0.08. 
(Other  details  arc  given  on  p.  519.) 

^'  Literature  on  the  chemistry  of  growth  given  bj'  Aron,  Handbuch  d.  Biochem., 
Ergiinzungsband,  1913. 

^^  Concerning  protamins,  see  rt'sum6  by  Kossel,  Biochem.  Centr.,  1906  (5), 
1  and  33. 

"  8{!e  also  (ireene,  Jour.  Biol.  Chcm.,  1919  (39),  435. 


PROLIFERATION  AND  REGENERATION  279 

eggs  synthesize  great  quantities  of  nucleoprotein,^*  even  when  in  a 
solution  free  from  phospliatcs,  and  hero  tlic  only  available  sourc(!  for 
the  phosphoric  acid  of  the  nucleins  would  seem  to  be  the  phospholipins 
of  the  egg  (J.  Locb).  The  nucleoproteins  during  karyokinesis  undergo 
a  chemical  change  in  that  they  become  of  a  more  acid  type  (presum- 
ably through  splitting  off  of  part  of  the  proteins  from  the  nucleic 
acid),  which  results  in  the  characteristic  increase  in  afh.nity  for  basic 
dyes,  and  the  increased  negative  charge  which  is  easily  demonstrated." 
This  suggests  the  participation  of  an  enzyme  in  the  process  of  karyo- 
kinesis, just  as  there  seems  to  be  in  the  production  of  pycnosis  in  de- 
generating cells,  but  there  seems  to  be  no  conclusive  evidence  on  this 
point.  Gies^^  could  find  no  enzyme  in  spermatozoa  that  incites  cell 
division  in  the  ova  of  sea-urchins  (Arbacia).  The  fertilization  of 
eggs  makes  them  more  permeable  to  ions,"  wliich  possibly  determines 
many  of  the  subsequent  changes. 

In  metaplasia  we  have  what  may  be  interpreted  as  a  chemical  alter- 
ation due  to  mechanical  stimuli,  e.  g.,  the  formation  of  keratin  by  cells 
that  ordinarily  do  not  do  so;  the  deposition  of  calcium  salts  and  oste- 
oid transformation  of  connective  tissues  in  rider's  bone,  etc.  That 
such  is  the  case,  however,  cannot  be  positively  stated  from  the  evidence 
at  hand. 

CHEMICAL  BASIS  OF  GROWTH  AND  REPAIR=8 

We  do  not  know  just  what  substances  are  necessary  to  maintain 
individual  cells  in  normal  condition,  what  are  needed  to  stimulate 
them  to  multiplication,  or  what  elements  they  require  to  permit  them 
to  multiply,  but  it  has  been  learned  that  certain  definite  materials  are 
required  by  the  organism  as  a  whole.  It  is  not  sufficient  that  a  given 
number  of  calories  with  a  certain  quantity  of  proteins,  carbohy- 
drates, fats  and  salts  be  supplied;  it  is  essential  that  certain  specific 
constituents  be  provided  among  these  foodstuffs.  This  fact  was  first 
clearly  pointed  out  by  Gowland  Hopkins  in  1906,  although  in  1897 
Eijkman  had  discovered  that  beriberi  and  experimental  neuritis 
might  result  from  a  one-sided  diet  of  polished  rice,  and  in  1902  Roh- 
mann  reported  that  purified  food  stuffs  do  not  suffice  to  maintain  and 
rear  mice. 

The  proteins  must  not  only  provide  a  sufficient  amount  of  nitrogen, 
but  they  must  also  provide  certain  specific  amino-acids,  as  has  been 
especially  demonstrated  by  the  investigations  of  Willcock  and  Hop- 

^*  Not  accepted  by  Masing,  Zeit.  physiol.  Chem.,  1910  (67),  161. 

"See  Gallardo,  Arch.  Entwickl.  Organ.,  1909  (28),  125;  Pentimalli,  ibid., 
1912  (34),  444. 

26Amer.  Jour.  Phvsiol.,  1901  (6),  54. 

"  See  AlcClendoii,  Carnegie  Inst.  Publ.,  1914.  No.  183. 

^^  See  Mendel,  "Nutrition  and  Growth,"  Harvey  Society  Lectures,  1914-15; 
Amer.  Jour.  Med.  Sci.,  1917  (153),  1;  Lusk,  "Science  of  Nutrition,"  Saunders, 
Phila.,  1917. 


280  THE  CHEMISTRY  OF  GROWTH  AXD  REPAIR 

kins^^  and  Osborne  and  Mendel.^"  Apparently  the  presence  of  some 
of  the  simple  straight-chain  amino-acids  can  be  dispensed  with  (e.  g., 
glycine),  and  the  animal  will  grow  and  thrive  if  other  nutritive  supplies 
are  adequate,  but  certain,  at  least,  of  the  more  complex  cj'clic  amino- 
acids  must  be  provided.  Furthermore,  the  requirements  for  growth 
(quantitatively  speaking  at  least),  seem  to  be  something  more  than 
the  requirements  for  mere  preservation  of  health  and  equilibrium, 
for  it  was  found  that  animals  could  live  and  preserve  nitrogen  equili- 
brium when  the  protein  of  the  diet  furnished  at  most  small  quantities 
of  lysine,  but  young  animals  were  unable  to  grow  with  such  a  restricted 
supply  of  this  amino-acid.  If  lysine  was  added  to  the  defective  pro- 
tein (gliadin  from  wheat)  the  animal  would  then  be  able  to  grow  at  a 
normal  rate.  Of  particular  importance  is  the  fact  that  animals  can 
be  kept  in  a  stunted  condition  on  such  a  deficient  diet  until  they  have 
reached  an  age  at  which  normally  all  growth  would  have  long  since 
ceased,  and  then  when  supplied  with  sufficient  lysine  they  will  begin  to 
grow  and  continue  until  full  size  is  reached. ^^  This  last  observation 
proves  that  growth  is  not  conditioned  by  age,  and  that  we  do  not  stop 
growing  because  a  certain  age  is  reached;  the  capacity  for  growth  may 
remain  latent  and  capable  of  exhibiting  itself  when  proper  conditions 
are  furnished.  But  no  amount  of  any  amino-acid  will  cause  a  fulh^ 
grown  animal  to  grow  any  more,  so  it  would  seem  that  the  capacity 
for  growth  becomes  extinguished  when  it  has  been  utilized  to  a  certain 
fixed  extent,  and  remains  potent  until  it  has  been  completelj'^  utilized. 
If  the  only  protein  furnished  contains  no  tryptophane  the  animal 
cannot  maintain  itself  and  slowly  loses  weight  until  it  dies,  unless 
tryptophane  is  supplied.  If  zein  from  corn,  which  yields  neither  ly- 
sine nor  tryptophane,  is  the  sole  protein,  then  the  animal  cannot  grow 
unless  both  lysine  and  tryptophane  are  added  to  the  diet.  So  too. 
pure  casein  is  not  adequate  to  maintain  growth  because  of  its  low 
content  in  cystine,  but  if  cystine  is  added  the  nutritive  value  is 
much  increased.  That  the  pure  isolated  amino-acids  can  meet  the 
deficiencies  when  added  to  the  imperfect  protein  ration,  demonstrates 
that  proteins  serve  for  food  as  amino-acids,  and  not  as  larger 
complexes. 

VITAMINES  OR  FOOD  HORMONES  AND  DEFICIENCY  DISEASES '- 

Not   only  must  the  proteins  present   certain   essential    chemical 
compounds  to  the  living  and  growing  organism,  but  also  an  adequate 

29  Jour.  Physiol.,  1906  (35),  88. 

^°  Series  of  papers  in  Jour.  Biol.  Cheni.,  1912,  et  seg. 

'Mour.  Biol.  Chem.,  1915  (23),  439. 

'2  See  "Report  on  the  Present  State  of  Knowledge  Concerning  Accessory  Food 
Factors  (Vitainines),  Special  Report  No.  38  National  Health  Insurance  Act, 
London,  1919;  "The  Newer  Knowledge  of  Nutrition,"  E.  V.  McCoUum,  New 
York,  1919;  Blunt  and  Wang,  Jour.  Homo  Economics,  1920  (12),  1;  also  Sympo- 
sium in  Jour.  Ainer.  Med.  Assoc,  1918  (71),  937. 


V  IT  AMINES  281 

supply  of  the  essential  inorganic  salts  and  certain  other,  as  yet  un- 
identified, substances  are  necessary  to  permit  of  maintenance,  growth 
and  repair.  It  has  long  been  recognized  clinically  that  certain  diseases, 
notably  scurvy,  may  result  from  the  absence  of  some  essential  in  the 
food  supply.  More  recently  other  diseases  have  been  proved  to  have 
a  similar  cause,  and  the  study  of  one  of  these,  beriberi,  has  led  to  a 
better  appreciation  of  the  nature  of  the  food  essentials  concerned. 
This  disease  seems  to  result  from  the  use  of  polished  rice  as  the  chief 
constituent  of  the  diet,  and  can  be  checked  by  feeding  unpolished  rice, 
or  rice  polishings,  or  even  extracts  of  rice  polishings,  as  first  demon- 
strated by  Eijkman.  A  somewhat  similar  condition  may  be  produced 
readily  in  birds  by  feecUng  them  only  polished  rice,  the  chief  feature 
being  a  severe  neuritis,  which  is  relieved  with  remarkable  rapidity 
by  supplying  the  food  deficiency.  This  experimental  neuritis  of  fowls 
{polyneuritis  gallinarum)  has  served  as  a  valuable  means  of  study  of 
diseases  of  this  class,  and  led  to  the  demonstration  that  not  only  ex- 
tracts of  rice  polishings,  but  also  many  other  food  materials,  contain 
the  essential  materials  without  which  health  cannot  be  maintained. 
One  of  the  early  investigators  of  this  subject,  Casimir  Funk,^^  gave 
to  "the  hitherto  unrecognized  essential  dietary  factors"  the  name  "vi- 
tamines,"  which,  in  spite  of  certain  logical  objections,  has  been  widely 
adopted;  but  as  Lusk  states,  the  term  "food  hormones"  would  be 
preferable  in  our  present  state  of  knowledge.  Although  so  essential 
for  life  the  amount  required  is  very  small,  for  whole  rice  is  said  to  con- 
tain not  over  0.1  gm.  per  kilo,  and  perhaps  much  less,  of  the  active 
substance. 

McCollum^*  has  summarized  the  evidence  that  two  classes  of 
substances  are  necessary  for  maintenance.  These  he  designated,  for 
convenience,  "/ai  soluble  A'"  and  '^water  soluble  B."  It  is  the  former 
that  is  lacking  in  xerophthalmia,  and  the  latter  in  poljmeuritis.  It 
now  seems  certain  that  other  diseases  are  the  result  of  deficiency  in 
other  specific  substances,  ^^  particularly  scurvy,  which  seems  to  result 
from  lack  of  a  "water-soluble  C."  It  also  is  an  open  question 
whether  under  the  water-soluble  B  are  included  two  separate 
vitamines,  one  antineuritic,  the  other  growth-promoting.^^" 

As  yet  the  exact  identity  of  the  active  agents  in  water  or  fat  solu- 
tions has  not  been  determined.  The  fat-soluble  vitamines  seem  to  be 
especially  abundant  in  butter,  egg  yolk,  and  cod  liver  oil,  which  pre- 
sumably accounts  for  the  commonly  accepted  values  of  these  partic- 
ular fats.  They  cannot  be  replaced  by  any  of  the  known  components 
of  fats,  including  phosphatids,  lipochromes,  cholesterol,  etc.,^^  and 

''  See  Ergeb.  Physiol.,  1913  (13 j,  125,  for  review  of  his  work. 
3"  Jour.  Biol.  Chem.,  1916  (24;,  491. 
3»  See  Jour.  Biol.  Chem,  1918  (33),  55. 
35»See  Mitchell,  Jour.  Biol.  Chem.,  1919  (40),  399. 

36  See  Drummond,  Biochem.  Jour.,  1919  (13),  81;  Palmer,  Science,  1919  (50), 
501. 


282  THE  CHEMISTRY  OF  GROWTH  AND  REPAIR 

are  scanty  or  absent  in  lard,  olive  oil,  and  most  vegetable  oils.  Funk 
believed  the  water-soluble  antineuritic  agents  to  be  pyrimidine  deriva- 
tives. They  are  dialyzable  (Drummond)  and  are  adsorbed  by 
Fuller's  earth  (Seidell).  Williams  and  Seidell"  have  found  that 
hydroxypurines  have  marked  anti-neuritic  effects,  and  they  sug- 
gested that  an  isomer  of  adenine-  is  responsible  for  the  anti-neuritic 
action  of  yeast  extracts.  Later  Williams^^  found  an  active  hydroxy- 
pyridene,  and  suggested  that  the  curative  properties  of  yeast  and 
rice  polishings  may  be  due  to  an  isomeric  form  of  nicotinic  acid.  These 
observations  await  confirmation,  and  we  still  are  in  the  dark  con- 
cerning the  character  of  antineuritic  vitamines.^^ 

The  nature  of  the ' '  fat-soluble  A  "  is,  if  possible,  even  less  known  than 
that  of  "water  soluble  B."  Drummond's  investigations^"  show  that 
it  is  somewhat  heat  resistant,  but  it  is  destroyed  at  100°  for  one  hour, 
apparently  not  through  oxidation  or  hydrolysis.  It  cannot  be  ex- 
tracted from  oils  by  water  or  dilute  acid,  but  is  extracted  to  some  ex- 
tent by  cold  alcohol.  If  the  fats  are  hydrolyzed  at  room  temperature 
the  active  factor  disappears,  and  it  cannot  be  identified  with  any  of 
the  recognized  components  of  fats.  Because  of  its  thermolability  and 
other  properties,  Drummond  is  driven  to  the  conclusion  that  ''fat- 
soluble  A"  is  not  a  clearly  defined  chemical  substance,  but  rather  it  is  a 
labile  substance,  perhaps  possessing  characteristics  resembling  those 
of  an  enzyme. ^"^^ 

Vitamines,  especially  those  that  are  water-soluble,  also  favor 
the  growth  of  bacteria, ^"^  and  are  essential  for  the  growth  of  yeast, 
so  that  Williams^^"  has  found  it  possible  to  determine  the  amount  of 
this  vitamine  present  in  a  food  stuff  by  the  rate  of  growth  of  yeasts 
thereon.  Typhoid  bacilli  are  said  to  produce  vitamines  during 
their  growth, ^'^'^  and  if  it  is  true,  as  has  been  stated,  that  neither 
plants  nor  animals  seem  able  to  synthesize  them,  it  would  seem  that 
they  must  be  of  bacterial  origin.  Yeast  is  known  to  produce  water- 
soluble  vitamine  in  particular  abundance,  but  not  the  fat- soluble 
vitamine.  Other  sources  of  water-soluble  vitamines  are  numerous, 
especially  green  vegetables  and  whole  cereals,  but  they  are  not  so 
abundant  in  meat  or  milk.^°« 

Why  the  vitamines  are  essential  and  how  they  act  is  unknown. 
It^s  suggestive  that  they  are  found  especially  in  cells  with  an  active 
metabolism,  but  whether  as  a  result  of  this  activity  or  because  essential 

"  Jour.  Biol.  Chem.,  1916  (26),  431. 
'«  Jour.  Biol.  Chem.,  1917  (29),  495. 

'^  See  review  by  Drummond,  Biochem.  Jour.,  1917  (11),  255. 
"»  liiochem.  Jour.,  1919  (13),  81. 

■"•"See  also  Steenbock  and  Boutwell,  Jour.  Biol.  Chem.,  1920  (41),  163. 
"''See  D.  J.  Davis,  Jour.  Infect.  Dis.,  1917(21),  392;  Kligler,  Jour.  Exp.  Med., 
1919  (30),  31. 

^'"^  Jour.  Biol.  Chem.,  1919  (38)  465;  also  Baehmann,  ibid.,  (39),  235. 

■""'  Pacini  and  llussell,  Jour.  Biol.  Chem.,  191S  (34),  43. 

"""See  Osborne  and  Mendel,  Jour.  Biol.  Chem.,  1919  (39),  29;  1920  (41),  515. 


i 


DKFICIKXCV  IJISKASES  283 

for  cell  growth  is  undeterminctl."  Voddor''-  has  suggested  that  Ihe 
aiiti-ncuritic  vitaniinc  is  essential  for  growth  and  repair  of  the  nervous 
tissue,  and  in  its  absence  normal  wear  cannot  be  made  good.'*'  There 
is  evidence  that  substances  rich  in  the  anti-neuritic  vitamine  stimulate 
growth  in  infants. ""^  Moore"*^  suggests  that  deficiency  diseases  may- 
be the  result  of  lack  of  something  needed  to  neutralize  toxic  sub- 
stances produced  in  metabolism  or  derived  from  outside  sources,  just 
as  in  poisoning  with  tri-nitro  toluene  and  similar  compounds  there  is 
no  intoxication  so  long  as  the  body  can  furnish  sufficient  glycuronic 
acid  to  neutralize  the  poisons.  Dutcher""  finds  some  relation  between 
catalase  and  vitamine  content  in  experimental  polyneuritis,  and  sug- 
gests that  the  vitamines  stimulate  oxidative  processes  which  remove 
toxic  substances.  It  is  probable  that  more  than  one  vitamine  is  neces- 
sary for  maintaining  normal  conditions,  and  deficiency  of  one  causes 
beriberi,  of  another  scurvy,  for  some  dietaries  lead  to  one  disease  and 
some  to  the  other.  Quite  possibly  minor,  or  less  well-defined  im- 
pairment in  health  may  often  result?  from  quantitative  deficiency  in 
vitamine  supplies.  The  main  points  concerning  the  most  studied 
deficiency  diseases  may  be  summarized  as  follows : 

Beriberi^^  occurs  in  two  forms — the  dry  polyneuritic  type  and  the 
edematous  or  wet  beriberi,  and  mixed  forms.  The  dry  type  resembles 
the  experimental  polyneuritis  of  birds,  mentioned  previously,  for  in  the 
birds  edema  does  not  accompany  the  polyneuritis  that  can  be  produced 
experimentally  by  feeding  polished  rice.  There  now  seems  to  be  no 
doubt  that  human  beriberi  is  the  result  of  the  absence  of  certain  es- 
sential elements  in  the  diet,  observed  especially  when  the  diet  is 
polished  rice,  but  possibly  occurring  with  other  deficient  diets,  for  the 
necessary  vitamine  is  of  course  present  in  many  other  foods  than  rice. 
Not  only  has  a  condition  closely  resembling  human  beriberi  been  pro- 
duced in  animals,  but  also  true  beriberi  has  been  experimentallj' 
produced  in  man  by  feeding  on  polished  rice,  as  well  as  the  repeated 
demonstration  of  both  prevention  and  cure  of  the  human  disease  by 
proper  feeding   or  by  administration  of  rice  polishings  or  extracts 

"  See  Voegtlin  and  Myers,  Amer.  Jour.  Physiol.,  1919  (48),  504. 

"  Jour.  Amer.  Med.  Assoc,  1916  (67 j,  1494. 

''^  McCarrison  has  observed  hypertrophy  of  the  adrenals  in  pigeons  with  experi- 
mental beriberi,  although  the  other  organs  are  atrophied.  He  considers  a  general 
nuclear  starvation  from  lack  of  necessary  nuclear  nutritive  materials  to  be  the 
essential  condition.  (India  Jour.  Med.  Res.,  1919  (6),  275.)  He  found  the- sex 
glands  particularly  atrophied,  and  Houlbert  (Paris  Medicate,  1919  (9;,  473,  has 
found  water-soluble  vitamines  essential  for  growth  of  sex  glands.  Emmett  and 
Allen  (Jour.  Biol.  Chem.,  Soc.  Proc,  1920  (41),  liii),  obtained  adrenal  hypertrophy 
with  thymus  atrophy  in  rats  fed  diets  deficient  in  water-soluble  B,  but  not  with 
diets  deficient  in  A.  Growth  of  tadpoles  also  seemed  to  be  more  accelerated  by 
B  than  bv  A. 

^*  Daniels  et  al,  .Amer.  Jour.  Dis.  Chil.,  1919  (18),  546. 

^^  British  Publ.  on  Munitions,  No.  11. 

«»  Jour.  Biol.  Chem.,  1918  (36),  6.34. 

*^  Full  discussion  and  bibliography  given  by  Vedder  in  his  book  "Beriberi," 
New  York,  1913.     See  also  Jour.  Amer.  Med.  Assoc,  1916  (67),  1494. 


284  THE  CHEMISTRY  OF  GROWTH  AND  REPAIR 

thereof.  From  rice  polishings  has  been  obtained  a  crystaUine  sub- 
stance, of  which  a  dose  of  20  to  30  milHgrams  will  cure  a  polyneuritic 
bird.  As  stated  above,  the  pure  active  substance  has  not  been 
isolated  and  its  exact  nature  is  undetermined.  Vedder  believes  that 
it  is  something  that  is  needed  for  the  repair  of  nervous  tissue,  so  that 
in  its  absence  the  nervous  tissues  degenerate.  The  paralysis,  he 
believes,  depends  more  on  central  than  peripheral-  nerve  changes, 
since  the  degeneration  of  the  nerves  precedes  the  paralj^sis  and  maj^ 
persist  long  after  the  paralysis  has  disappeared.  As  rice  polishings 
relieve  the  cardiac  symptoms,  which  are  important  features  of  beri- 
beri, it  is  to  be  assumed  that  the  vitamine  is  essential  for  the  heart 
metabolism.  Furthermore,  heart  muscle  contains  vitamine  which 
will  protect  from  polyneuritis  birds  fed  on  polished  rice.  This  does 
not  seem  to  be  identical  with  the  vitamine  isolated  by  Funk,  for 
while  it  relieves  the  cardiac  symptoms  and  dispels  the  dropsy  of  wet 
beriberi,  it  does  not  cure  the  paralytic  symptoms  of  dry  beriberi, 
according  to  Vedder.  This  author  "has  a  growing  belief  that  dry 
and  wet  beriberi  are  separate  and  distinct  diseases,  which  are,  how- 
ever, generally  associated."  Rice  polishings,  he  says,  clear  up  beri- 
beri dropsy  quickly,  but  do  not  affect  the  paralysis  unless  the 
polishings  have  been  hydrolyzed. 

Walshe^^  calls  attention  to  the  fact  that  starved  fowls  live  long 
enough  to  develop  beriberi,  yet  nevertheless  do  not  show  it,  so  he 
thinks  that  there  must  not  only  be  a  deficiency  factor,  but  also  some 
positive  factor,  which  may  be  the  abundant  carbohydrate  of  the 
rice  diet.  Possibly  in  the  absence  of  the  vitamine  the  carbohydrate 
metabolism  is  altered,  with  the  production  of  toxic  substances. 
Furthermore,  in  spite  of  the  marked  clinical  results  obtained  with 
rice  polishings,  the  disease  is  not  always  cleared  up  as  readily  as 
might  be  expected  if  only  a  lack  of  vitamines  was  concerned,  and, 
therefore,,  there  still  remains  the  possibility  that  some  infectious 
factor  may  play  at  least  a  subsidiary  part  in  human  beri-beri  (see 
Mitchell  3^''). 

Keratomalacia  or  Xerophthalmia,'^  a  condition  of  opacity  of 
the  cornea,  followed  by  ulceration  and  blindness,  seems  to  be  specifi- 
cally due  to  lack  of  the  fat-soluble  vitamine  which  is  present  in  egg 
yolk,  butter  fats,  green  leaves,  etc.,  but  not  in  lard  or  in  many  vege- 
table oils.  This  disease  can  be  produced  readily  in  experimental 
animals  by  feeding  diets  free  from  proper  fats,  and  is  relieved  by 
administration  of  small  quantities  of  these  fats.  I  have  had  the 
opportunity  to  observe  numerous  instances  of  xerophthalmia  among 
the  famine  sufferers  in  Roumania,  and  to  observe  its  prompt  relief 
under   cod  liver  oil  feeding.     It  should  not  ha  confused  with  simple 

"  (iuart.  Jour.  Med.,  1918  (11),  320. 

^8  Bloch,  Ugeskr.  f.  Laeger.,  1918  (80),  815. 


DEFICIENCY  DISEASES  285 

eye  infections  wliicli  arc  likely  to  occur  in  poorly  nourished  laboratory 
animals/^ 

Nutritional  Dropsy.     ("War  Dropsy"  or  Famine  Edema). "^^ 

This  condition,  which  was  observcul  extensively  durinj^  the  war, 
especially  among  Russian  prisoners  in  Germany,  has  been  seen  wherever 
famine  occurs,  and  is  undoubtedly  caused  bj^  dietary  deficiency. 
Apparently  it  is  independent  of  scurvy.  As  it  is  often  associated  with 
xerophthalmia  it  has  been  thought  to  depend  on  al)sence  of  fat-soluble 
vitamines.  It  seems  more  probable,  however,  that  it  results  from 
low  caloric  supply,  although  protein  deficiency  combined  with  exces- 
sive fluid  and  salt  intake  in  the  effort  to  maintain  life  with  weak  soups, 
are  probably  important  factors.^'  Most  of  the  adult  subjects  have 
been  receiving  800  to  1200  calories  per  day,  containing  })ut  30  to  50 
gms.  of  protein.  It  is  much  more  likely  to  appear  in  undernourished 
persons  who  are  compelled  to  work  than  in  equally  starved  persons  at 
rest,  since  work,  and  also  cold,  increase  the  caloric  deficiency. 

Numerous  studies  of  metabolism  and  blood  chemistry  in  persons 
exhibiting  war  dropsy  have  given  concordant  results,  which  show  the 
extreme  depletion  of  the  body  in  all  nutritive  reserves.*^  The  blood 
shows  hypoglucemia,  decrease  in  potassium,  fatty  acids,  phospholipins 
and  residual  nitrogen,  with  anincrease  in  NaCl,  and  of  acetone  bodies 
and  ammonia  from  starvation  acidosis.  There  is  also  a  decrease  in 
the  amount  of  protein  in  the  blood  even  to  one-half  the  normal  amount, 
with  hj^dremia  and  a  less  marked  decrease  in  both  red  and  white  cells. 
The  lack  of  reserve  nitrogenous  material  both  in  blood  and  tissues  is 
shown  by  the  fact  that  when  patients  with  war  edema  are  fasted 
a  few  days  the  N  excretion  may  fall  to  2  to  3  gms.  per  day,  while  in 
absolute  starvation  of  previously  normal  persons  the  N  output  usually 
is  10-12  gms.  (Falta).  Schittenhelm  and  Schlecht  suggest  that  the 
edema  is  merely  the  result  of  injury  to  the  capillary  endothelium,  in 
common  with  all  the  other  tissues,  whereby  their  permeability  becomes 
increased. 

Famine  edema  seems  to  be  closely  related  to  the  edema  often 
observed  in  infants  kept  on  a  preponderatingly  starchy  diet,  such  as 
•  barley  water,  for  long  periods.  Here  most  striking  degrees  of  dropsy 
are  observed,  which  seem  in  all  respects  similar  to  famine  dropsy. 
There  is  probablj^  also  a  close  relation  to  the  edema  of  pernicious 
anemia   and  cachexia.     The  relation  of  this  form  of  edema  to  the 

"  See  Bulley,  Biochem.  Jour.,  1919  (13).  103. 

"Full  Review  by  Schittenhelm  and  Schlecht,  Zeit.  exp.  Med.,  1919  (9),  1; 
Maver,  .lour.  Amer.  Med.  Assoc,  1920  (74),  934. 

^'  See  Guillermine  and  Guyot,  Rev.  Med.  Suisse  Rom.,  1919  (39),  115;  Falta, 
Wien.  klin.  Woch.,  1917  (30),'  1637;  Schittenhelm  and  Schlecht,  Zeit.  exp.  Med., 
1919  (9),  82. 

"See  Schittenhelm  and  Schlecht,  loc.  cit.;  Feigl,  Biochem.  Zeit.,  1918  (85), 
365;  Jan&en,  Miinch.  Med.  W'ocK,  1918  (65),  925;  Biirger,  Zeit.  exp.  Med.,  1919 
(8),  309. 


286  THE  CHEMISTRY  OF  GROWTH  AND  REPAIR 

edema  of  wet  beriberi  has  not  been  determined,  but  it  is  highly  prob- 
able that  their  origin  has  something  in  common.  Both  are  dropsies 
due  to  diet  deficiency,  and  it  may  well  be  that  the  deficiency  is 
the  same  in  each  case.  In  both  these  conditions,  as  well  as  in  the 
"Mehlnahrschaden"  of  starch-fed  babies,  there  is  the  common  ele- 
ment of  relatively  excessive  carbohydrate  supply,  which  may  have 
something  to  do  with  the  dropsy.  The  clinical  evidence  is  against  the 
view  that  nutritional  edema  depends  on  a  lack  of  specific  vitamines.^-" 

Experimental  work  supports  the  clinical  evidence  as  to  the  etiology 
of  nutritional  edema.  Miss  Kohman^^  has  found  that  rats  fed  diets 
composed  chiefly  of  carrots  often  develop  a  severe  edema,  which  is 
prevented  by  supplying  protein,  but  not  by  butter  fat  or  starch. 
Evidently  neither  fat-soluble  nor  water-soluble  vitamines  are  respons- 
ible. It  was  found  that  on  a  dry  diet  of  equal  caloric  and  protein  de- 
ficiency the  rats  are  not  so  likely  to  develop  edema.  Experiments 
done  in  my  laboratory  by  M.  B.  Maver"^"  agree  fully  with  those  of  Miss 
Kohman.  Apparently  low  protein  and  high  fluid  intake  are  the  most 
essential  factors,  although  relatively  high  carbohydrate  must  also  be 
considered. 

Scurvy  would  seem  almost  certainly  to  be  a  deficiency  disease, 
but  there  has  been  much  disagreement  as  to  this  point,  especially 
among  those  who  have  studied  experimental  scurvy  in  animals.  There 
is  room  for  doubt  that  the  expei'imental  disease  in  animals  is  identical 
with  human  scurvy,  at  least  there  is  reason  to  believe  that  more  than 
one  concHtion  has  been  described  as  scurvy  in  experimental  animals. 
Apparently  guinea  pigs,  however,  develop  readily  a  disease  which 
resembles  scurvy  very  closely  both  anatomically  and  in  its  relation  to 
dietary  conditions.  Hess,  who  has  studied  especially  infantile  scurvy, 
finds  that  orange  juice  given  intravenously  will  relieve  scurvy,  and 
thus  apparently  disposes  of  all  theories  of  gastrointestinal  disorders  as 
the  responsible  factor.  Artificial  "orange  juice,"  (containing  sugar, 
citric  acid  and  inorganic  salts  in  the  proportions  found  in  natural 
orange  juice)  is  ineffective,  so  that  apparently  scurvy  is  the  result  of 
lack  of  some  undetermined  substance  present  in  orange  juice  as  well 
as  in  other  fresh  vegetable  foods.  As  yet  we  have  no  evidence  as  to 
the  character  of  this  "vitamine,"  which  has  been  designated  as 
"water-soluble  C,"  but  it  is  probably  quite  distinct  from  either  water- 
soluble  B  or  fat-soluble  A,^^  and  Hess  believes  that  our  ordinary 
dietary  probably  does  not  contain  any  great  excess  of  the  antiscorbutic 
element,  since  scurvy  so  readily  appears  when  the  necessary  vegetable 
foods  arc  reduced  in  amount.  According  to  Chick  and  Hiune  this 
vitaminc  is  present  in  living  vegetable  and  animal  tissues,  in  largest 

«2«See  Burger,  Zeit.  Exp.  Med.,  1919  (8),  309. 

"  Denton  and  Kohman,  Jour.  Biol.  Chem.,  1918  (36),  249;  Kohman,  Amer. 
Jour.  Physiol.,  1920  (51),  378. 

"Cohen  and  Mendel,  Jour.  Biol.  Chem.,  191S  (35),  425. 


,•-»' 


DEFICIENCY  DISEASES  287 

amounts  in  fresh  fruits  and  grcrn  vegetables,  to  a  less  extent  in  root 
vegetables  and  tubers.  It  is  present  in  small  amount  in  fresh  moat 
and  milk,  and  has  not  3'et  been  detected  in  yeast,  fats,  cereals,  pulses. 
The  explorer,  Stefansson,^^  has  reported  observations  indicating  the 
presence  of  antiscorbutic  substances  in  raw  meat,  and  their  absence 
or  deficiency  in  well-cooked  meat  and  tinned  foods.  Evidently  this 
antiscorbutic  element  is  very  unstable,  since  even  drying  vegetables 
at  moderate  temperatures,  6.5-70°,  and  cooking  or  salting  meats,  or 
heating  with  weak  alkalies,  destroys  or  greatly  reduces  their  antiscor- 
butic value. ^^  Pasteurization  of  milk  also  reduces  the  preventive 
value  of  this  food"  which,  in  its  raw  state,  contains  in  abundance 
all  necessary  factors  for  nutrition,  but  apparently  little  more  of  the 
antiscorbutic  substance  than  is  barely  sufficient  to  maintain  health. 

Pellagra^^  probablj-  belongs  among  the  deficiency  diseases,  despite 
numerous  attempts  to  account  for  it  as  an  infectious  disease.  The 
work  of  Goldberger^^  is  especially  valuable  in  affirmative  evidence 
of  the  relation  of  dietary  deficiency  to  pellagra.  Here  again  we  are 
entirely  uninformed  as  to  the  nature  of  the  deficiencj'.  Goldberger®'' 
sums  up  his  conclusions  as  follows:  "The  pellagra-producing  dietary 
fault  is  the  result  of  some  one,  or,  more  probably,  of  a  combination  of 
two  or  more  of  the  following  factors:  (1)  a  physiologically  defective 
protein  supply;  (2)  a  low  or  inadequate  supply  of  fat-soluble  vitamine; 
(3)  a  low  or  inadequate  supply  of  water-soluble  vitamine,  and  (4)  a 
defective  mineral  supply.  The  somewhat  lower  plane  of  supply,  both 
of  energy  and  of  protein,  of  the  pellagrous  households,  though  appa- 
rently not  an  essential  factor,  ma}',  nevertheless,  be  contributory  by 
favoring  the  occurrence  of  a  deficiency  in  intake  of  some  one  or  more 
of  the  essential  dietary  factors,  particularlj^  with  diets  having  only  a 
narrow  margin  of  safety.  The  pellagra-producing  dietary  fault  may 
be  corrected  and  the  disease  prevented  by  including  in  the  diet  an 
adequate  supply  of  the  animal  protein  foods,  particularly  milk,  in- 
cluding butter  and  lean  meat. " 

McCollum  calls  attention  to  the  fact  that  a  diet  is  not  adequate 
unless  it  contains  active  metabolizing  protoplasm,  as  found  in  green 
leaves,  eggs,  meat  and  milk;  and  pellagra-producing  diets  are  largely 
composed  of  seed  foods  and  pork  fat.  To  make  cereal  grains  diete- 
tically  satisfactory  there  must  be  added  inorganic  elements,  a  protein, 
and  substances  containing  "fat  soluble  A."^' 

"  Jour.  Amer.  Med.  Assoc,  1918  (71),  1715. 

"  Givens  and  Cohen,  Jour.  Biol.  Chem.,  1918  (36),  127;  Amer.  Jour.  Dis.  Chil., 
1919  (18),  .30. 

*'  See  Hess,  Amer.  Jour.  Dis.  Chil.,  1919  (17),  221. 

'*  Concerning  metabolism  in  pellagra  see  ]\Iyers  and  Fine,  Amer.  Jour.  Med. 
Sci.,  1913  (145)  705.  Chemical  changes  in  the  central  nervous  system  described 
by  Koch  and  Voegtlin,  Hygienic  Lab.  Bull.  103,  1916. 

"Public  Health  Rep.,' 1914  (29),  1683;  1915  (30),  3117,  3336. 

«»  Jour.  Amer.  Med.  Assoc,  1918  (71),  944. 

«>  Jour.  Biol.  Chem.,  1919  (38),  113. 


288  THE  CHEMISTRY  OF  GROWTH  AND  REPAIR 

Whatever  the  deficiency  in  diet  may  be,  pellagra  seems  to  develop 
most  often  in  persons  whose  diet  is  preponderatingly  maize  seed 
products.  Only  in  countries  where  maize  is  the  chief  dietary  staple 
does  pellagra  occur  with  any  great  frequency,  and  in  those  countries 
where  part  of  the  population  lives  chiefly  on  maize,  and  other  groups 
live  on  other  foods,  pellagra  occurs  chiefly  or  only  in  the  former  group. 
I  have  had  the  opportunity  to  observe  much  pellagra  in  Roumania 
during  a  period  of  protracted  and  serious  food  shortage,  and  this 
relation  to  maize  was  most  striking  and  convincing.  The  peasants 
of  this  country  have  for  their  cliief  food  a  thick  mush  of  boiled,  coarsely 
ground  corn  meal,  called  mamaliga,  supplemented  by  such  other  foods 
as  they  can  secure.  Dwellers  in  the  towns  rely  on  bread  from  wheat 
flour  as  their  chief  carbohydrate  supply,  and  have  a  much  more  abun- 
dant and  varied  list  of  accessory  foods.  Pellagra  is  prevalent  in  Rou- 
mania, but  restricted  to  the  maize-eating  peasants,  and  in  very  definite 
relation  to  their  inability  to  secure  accessory  food  stuffs.  While 
Roumanian  physicians  seem  generally  inclined  to  accept  the  theory 
that  spoiled  maize  is  responsible,  my  own  observations  would  indicate 
that  the  chief  difficulty  is  lack  of  accessory  foods.  The  relation  of 
maize  to  pellagra  becomes  particularly  striking  if  we  compare  Rou- 
mania with  a  country  where  maize  is  not  a  staple  food,  such  as  Korea. 
Here  for  centuries  a  large  part  of  the  population  has  existed  on  the 
verge  of  starvation,  the  chief  food  being  rice.  Although  here  beriberi 
is  common  enough,  especially  among  those  who  can  afford  the  luxury 
of  polished  rice,  pellagra  is  not  observed,  despite  a  much  greater 
deficiency  in  total  food  supply,  both  as  regards  calories  and  acces- 
sories, than  prevails  in  Roumania. 

Despite  the  abundant  evidence  of  the  relation  of  dietary  deficiency 
there  are  those  who  interpret  existing  evidence  as  establishing  or 
making  probable  that  pellagra  is  nevertheless  essentially  an  infectious 
disease. ^^  The  compromise  view  that  pellagra  is  an  infectious  disease 
which  can  only  manifest  itself  among  those  suffering  from  dietary 
deficiency  has  also  been  supported,  especially  by  INIcCollum.''^ 

Rickets. — Mellanby^^  holds  that  this  disease  results  from  a  defi- 
ciency in  fat-soluble  vitamine,  although  admitting  that  the  efficiency 
of  malt  extracts  and  lean  meat  in  preventing  experimental  rickets  is 
not  in  harmony  with  this  hypothesis.  As  the  total  growth  of  rachitic 
puppies  on  a  diet  poor  in  fat-soluble  A  is  about  normal,  he  suggests 
that  this  agent  is  not  necessary  for  growth,  but  merely  for  making 
growth  normal.  Without  "A"  the  development  of  teeth  is  much 
interfered  with.  The  recognized  value  of  cod  liver  oil  in  rickets  is  in 
support  of  this  view.  Hess,  McCollum  and  others  do  not  accept  the 
hypothesis  that  rickets  is  solely  the  result  of  lack  of  fat-soluble  A. 

"  See  Jobling  and  Peterson,  Jour.  Infect.  Dis.,  191G  (IS),  501. 

"  Proc.  Amer.  Philos.  Soc,  1919  (58),  41. 

«' Jour.  Physiol.,  1919  (.52),  liii;  Lancet,  1919  (19G),  407. 


DEFICIENCY  DISEASES  281) 

The  former  has  observed  children  who  have  hved  a  long  time  in  per- 
fect health  on  a  diet  with  a  minimum  of  vitamine-containing  fats.*° 
McCollum  finds  that  rats  develop  a  condition  apparently  identical 
with  true  rickets  when  kept  on  a  diet  deficient  in  any  two  of  the  three 
essentials,  viz.,  protein,  calcium,  fat-soluble  A.  A  diet  lacking  only 
one  essential  seems  to  be  well  borne. 

"Jour.  Amer.  Med.  Assoc,  1920  (74),  217. 


CHAPTER  XIII 

DISTURBANCES  OF  CIRCULATION  AND  DISEASES 
OF  THE  BLOOD 

THE  COMPOSITION  OF  THE  BLOOD 

The  function  of  the  blood  being  to  maintain  an  equilibrium  in 
the  temperature,  chemical  composition  and  osmotic  pressure  between 
all  parts  of  the  body,  it  follows  that  it  is  never  of  exactly  the  same 
composition  in  any  two  places  or  at  any  two  times.  To  the  extent 
that  every  tissue  is  continually  giving  off  something  to  the  blood,  we 
may  consider  that  every  organ  is  a  factor  in  its  formation,  and  as  a 
result  of  this  multiplex  origin  of  the  blood,  the  substances  it  may  con- 
tain are  beyond  enumeration.  There  are  probably  but  few  chemical 
substances  occurring  in  the  tissue-cells  that  do  not  also  occur  in 
greater  or  less  amount  in  the  blood.  In  addition  to  these  there  are 
also  the  substances  characteristic  of  the  blood  itself,  besides  a  host 
of  substances  of  unknown  nature,  apparently  manufactured  in  re- 
sponse to  the  stimulation  of  substances  entering  the  body  from  out- 
side; for  we  find  that  the  blood  of  every  adult  individual  contains 
substances  that  make  him  immune  to  a  multitude  of  diseases  that  he 
has  had  in  childhood,  as  well  as  substances  that  in  later  life  protect 
him  to  a  greater  or  less  degree  from  infection  by  such  organisms  as 
the  colon  bacilli  of  his  intestine,  the  pneumococci  and  streptococci 
in  his  throat,  etc.  We  have  learned  of  these  defensive  substances 
within  very  recent  times,  and  also  of  the  "antienzymes"  that  possi- 
bly protect  the  blood  from  the  digestive  enzymes  of  the  body  cells. 
What  other  substances  of  importance  we  may  yet  find  in  the  blood  is 
an  open  question.  There  are  no  apparent  limits  to  the  possibilities 
of  the  study  of  the  blood,  for  it  represents  a  little  of  every  organ,  and 
much  that  is  characteristic  of  itself. 

In  discussing  briefly  the  substances  that  have  been  isolated  from 
the  normal  blood,  before  considering  the  changes  that  occur  in  it 
during  pathological  conditions,  we  may  roughly  divide  the  blood  into 
the  formed  elements  and  the  plasma  in  which  they  are  suspended. 

THE  FORMED  ELEMENTS.— By  weight,  tlie  red  corpiu«clos  constitvito  from 
40  to  50  por  cent,  of  tli(>  l)l()0(l,  tlie  i)crccnt:i}i;c  varyinji;  uiiiUn-  (lilTc-ront  conditions, 
while  llie  total  weif^ht  of  the  leucocytes  and  platelets  is  insi<;iuticant.  'I'he  henio- 
fr|ol)in  constitutes  troni  Sti  to  94  i)er  cent,  by  \veit;;ht  of  tiie  solids  of  the  red  cor- 
puscles, but  the  i)liysical  and  chemical  relations  that  it  bears  lo  the  stroma  of  the 
corpuscles  are  as  yet  undetermined  (see  "Hemolysis").     t)f  tlie  remaining  constit- 

290 


COMPOSITION  OF  BLOOD  '291 

uents  of  the  corpuscles,  from  5  to  12  ywr  cent,  consist  of  proteins,  i)robal)ly  chiefly 
globulins  and  nuclcoprolcins;  0.3  to  0.7  per  cent,  of  lecithin;  and  about  0.2  to  0.3 
per  cent,  of  cholesterol  (Iloppe-Seyler).  The  outer  coat  of  the  red  corpuscles 
docs  not  seem  to  be  equally  permeable  for  all  substances,  and  therefore  we  find 
the  composition  of  the  fluid  p(jrtion  of  the  cell  quite  different  from  that  of  the 
plasma  about  it.  The  salts  of  tlie  corpuscles  consist  largely  of  potassium  pho.s- 
phate,  a  little  sodium  chloride,  some  magnesium,  but  no  calcium,'  which  is  quite 
different  from  their  proportion  in  the  plasma.  Probably  many  of  the  other  con- 
stituents of  the  plasma,  especially  urea,  ])enetrate  the  red  corpuscles  to  a  greater 
or  less  degree,  but  most  of  them,  particularly  the  sugar,  remain  chiefly  in  the 
plasma. 

Hemoglobin,  the  most  characteristic  constituent  of  all  the  heterogeneous  com- 
ponents of  the  blood,  is  a  compound  protein,  and  probably  exists  combined  with 
some  other  constituent  of  the  corpuscle,  most  probably  the  lecithin.  It  splits 
up  readily  into  a  protein,  glohin,  and  an  iron-containing  substance,  hemochromo- 
gcn,  which  readily  takes  up  oxygen  to  form  hematin.  Only  about  4  to  5  per  cent, 
of  the  hemoglobin  is  hemochromogen,  and  iron  constitutes  but  about  0.4  per  cent. 
Hematin  may  be  further  split  up  into  other  substances,  which  will  be  considered 
in  the  discussion  of  "Hemorrhage." 

The  leucocytes  consist  chiefly  of  nucleoproteins,  with  probably  some  globulin, 
and  they  also  contain  glycogen,  phospholipins,  and  cholesterol.  The  blond-platelets 
are  believed  to  be  largely  nucleoprotein,  but  little  is  known  of  their  actual  composi- 
tion; microchemical  examination  shows  no  evidence  of  either  fat  or  glycogen.* 

BLOOD  PLASMA  differs  from  blood-serum  in  that  the  latter  is  formed  from  the 
former  through  the  removal  of  the  fibrinogen  through  its  conversion  into  fibrin. 
Serum,  therefore,  contains  no  fibrinogen,  but  more  fibrin  ferment;  otherwise  it 
is  practically  the  same  as  the  plasma.^  It  is  well  for  us  to  appreciate  that  the  blood 
is  fundamentally  a  tissue,  with  its  more  solid  structural  elements  lying  in  a  pro- 
toplasm, the  plasma,  somewhat  more  dilute  than  the  protoplasm  of  other  tissues 
but  in  other  respects  much  the  same. 

Proteins. — Fibrinogen  has  the  general  properties  of  a  globulin,  with  also  a 
peculiar  tendency  to  go  into  the  insoluble  form,  fibrin.  (This  process  will  be 
discussed  under  "Thrombosis.")  In  the  plasma  are  also  other  globulins,'*  one 
soluble  in  water  (pseudo-globidin),  the  other  insoluble  in  water  {euglobulin) . 
Serum-albumin,  another  protein  of  the  plasma,  probably  consists  of  two  or  more 
varieties  of  albumin.  There  are  also  nucleoproteins  {prothrombin)  and  non- 
coagulable  proteias,  which  being  poorly  understood  have  been  variously  considered 
as  glycoproteins,  or  mucoids,  or  albumoses.  The  serum  proteins  seem  to  be  closely 
related  to,  or  compounded  with,  the  lipins  of  the  plasma. 

Other  Constituents. — The  fat  of  the  plasma  varies  much  according  to  the  time 
which  has  elapsed  after  the  taking  of  food;  in  fasting  animals  it  amounts  to  from 
0.1  to  0.7  per  cent.  The  sugar  fluctuates  less,  being  normally  about  0.1  per  cent., 
whUe  the  urea  has  been  estimated  at  0.03  per  cent.  Most  of  the  sugar  is  dex- 
trose; but  probablj'  there  is  some  levulose,  possibly  some  pentose  and  other  forms, 
and  possibly  also  sugar  combined  with  lecithin  (jccorin)  or  other  substances. 
Soaps,  cholesterol,  and  phospholipins  exist  free  in  the  plasma.  There  are  also  the 
numerous  nonprotein  nitrogenous  substances  that  are  excreted  in  the  urine. 

Plasma  differs  strikingly  from  the  corpuscles  in  that  its  inorganic  substances 
are  chiefly  sodium  and  chlorine,  while  potassium  and  phosphoric  acid  are  almost 
entirely  absent.  Another  important  fact  is  that  when  the  plasma  is  combusted,, 
the  acid  radicals  remaining  do  not  suffice  to  balance  the  bases,  indicating  that 
much  of  the  inorganic  bases  is  joined  with  organic  substances,  probabh'  as  ion- 

'  The  current  statement  that  corpuscles  are  impermeable  for  calcium  is  refuted 
by  Hamburger  (Zeit.  physikal.  Chem.,  1909  (69),  663). 

-  Aynaud,  Ann.  Inst.  Pasteur,  1911  (25),  56. 

^  In  the  process  of  clotting  certain  changes  occur,  probablj^  physical,  that  may 
make  the  plasma  more  or  less  toxic  (see  Anaphyla.xis)  and  apparently  alter  its 
biological  properties,  since  the  reinjection  of  a  person's  own  defibrinated  serum 
may  cause  marked  physiological  and  therapeutic  effects  {e.  g.,  autoserotherapy 
in  psoriasis).  Especially  noteworthv  is  the  vasoconstrictor  effect  of  defibrinated 
blood  (see  Hirose,  Arch.  Int.  Med.,  1918  (21),  604). 

*  Literature  given  by  Rowe,  Arch.  Int.  Med.,  1916  (18),  455. 


292  DISTURBANCES  OF  CIRCULATION 

protein  compounds.     The  alkali  joined  to  the  protein  is  non-diffusible,  and  con- 
stitutes about  five-sixths  of  the  total  alkali. 

The  concentration  of  the  electrolytes  of  the  blood  has  been  determined  by 
ascertaining  the  lowering  of  the  freezing-point,  which  in  human  blood  averages 
about  0.526°;  this  corresponds  closely  to  the  effect  of  a  salt  solution  of  0.9  per 
•cent,  strength.  About  three-fourths  of  the  dissolved  molecules  of  the  blood-serum 
are  electrolytes,  and  about  three-fourths  of  these  are  molecules  of  NaCl,  most 
of  which  are  in  the  dissociated  state.^  The  calcium  content  is  very  constant,  about 
9  to  11  mg.  per  100  cc.  of  plasma. 

Enzymes. — A  large  number  of  enzymes  exist  in  the  blood,  the  following  being 
among  those  that  have  been  detected:  diastase,  glucase,  lipase,  thrombin,  rennin, 
and  'proteases.  The  proteases  and  perhaps  the  other  enzymes  are  held  in  check 
to  a  large  extent  by  " antiferments"  that  are  also  present  (see  "Enzymes")-  In 
relation  to  the  antiferments  are  the  innumerable  antibodies  that  exist  normally 
in  the  serum  for  foreign  proteins,  foreign  cells,  and  for  bacteria  and  their  toxins, 
as  well  as  those  resulting  from  reaction,  etc. 

The  proportions  in  which  the  constituents  of  the  plasma  normally  occur  have 
been  determined  by  Hoppe-Seyler  and  by  Hammarsten,  as  follows:* 

Table  I 

No.  1  No.  2 

Water 908.4  917.6 

SoUds 91.6  82.4 

Total  proteins 77 . 6  69 . 5 

Fibrin 10.1  6.5 

Globulin 38.4 

Seralbumin 24 . 6 

Fat 1.2 

Extractive  substances 4.0 

Soluble  salts 6.4  12.9 

Insoluble  salts 1.7 

No.  1  is  an  analysis  by  Hoppe-Seyler. 

No.  2  is  the  average  of  three  analyses  made  by  Hammarsten. 

Reaction. — If  we  titrate  the  blood  plasma  with  an  acid,  we  liberate  much  of 
the  alkali  from  the  proteins,  dissociate  all  the  Na2C03  present,  as  well  as  the 
NaHCOs  and  the  sodium  phosphate,  and  find  in  this  way  that  the  entire  fresh 
blood  contains  neutralizable  alkali  corresponding  tc  a  solution  of  Na^COs  of  about 
0.443  per  cent,  strength  (Strauss;.  In  other  words  the  blood  has  a  quantity  of 
alkali  in  combination  that  can  be  drawn  upon  to  neutralize  acids  to  the  extent 
indicated  by  the  above  figures.  The  real  alkalinity  of  a  fluid,  however,  is  dependent 
upon  the  number  of  free  OH  ions  in  the  solution;  and  Hober  has  determined  by 
physico-chemical  methods  that  the  concentration  of  OH  ions  in  blood  is  but  little 
greater  than  in  distilled  water.''  Michaelis*  has  found  the  H+  concentration  of 
the  blood  to  be  0.45  X  10"',  as  contrasted  with  neutrality  at  38°  which  is  H4-  = 
1.5  X  10~'.  The  interchange  between  COo,  phosphates  and  carbonates  in  the 
blood  is  such  that  it  is  impossible  for  any  considerable  quantities  of  free  H  or 
OH  ions  to  exist,  and  the  protoplasm  is  thus  protected  from  an  excess  of  either. 
The  capacity  of  the  blood  to  neutralize  acids  and  alkalies  is  sometimes  referred  to 
as  its  "buffer  value."^  According  to  Henderson'"  not  more  than  five  parts  of 
excess  free  H  or  OH  ions  can  be  present  in  ten  billion  parts  of  protoplasm.  An 
alkalinity  is  impossible  because  this  would  cause  an  increased  osmotic  pressure  which 
the  kidneys  would  regulate;  acidity  is  impossible  because  death  would    result 

^  Concerning  relation  of  conductivity  to  freezing-point  see  Wilson,  Anier.  Jour, 
of  Physiol.,  1906  (16),  438. 

'^  For  complete  analyses  of  the  blood  see  Abderhalden,  Zeit.  physiol.  Chem., 
1S9.S  (25),  106. 

'  For  bibliography  on  Alkalinity  of  Blood,  see  Henderson,  Ergcbnisse  Physiol., 
1909  (8),  254. 

8  Deut.  med.  Woch.,  1914  (40),  1170. 

"  See  Levy  and  Rowntree,  Arch.  Int.  Med.,  1916  (17),  525. 

»"  Amer.  Jour.  Physiol.,  1907  (18^.  250;  1908  (21;,  427. 


IlKMOh-h'/fACK  29;i 

from  the  inability  of  the  blood  to  carry  CO2.  The  blood  and  tissue  jjrotcinf 
also  can  bind  much  of  either  H  or  OH  ions,"  so  that  the  preservation  of  neutrality 
is  elaborately  guarded.  In  the  tissues,  because  of  the  profluc.tion  of  acids  during 
metabolism,  the  Il-ion  concentration  is  slightly  higher  than  that  i)f  the  blood,  being 
estimated  by  Michaelis  at  exact  neutrality,  1.5  X  10"'.  Presumably  one  impor- 
tant purpose  of  the  e.xact  regulation  of  reaction  is  to  provide  proper  conditions  for 
enzyme  action. 

The  alkali  of  the  blood  exists  in  part  as  alkaline  salts,  carbonate  and  phosphate 
(the  diffusible  alkali),  and  partly  combined  with  protein  {non-diffusible  alkali). 
As  the  corpuscles  are  richer  in  diffusible  alkali  than  the  plasma  or  serum,  the  num- 
ber of  corpuscles  modifies  the  alkalinity  of  the  blood  decidedly.  Much  importance 
is  attached  to  the  question  of  the  alkalinity  of  the  blood  for  two  reasons:  first,  in 
certain  conditions  of  disease  the  blood  contains  so  much  of  organic  acids  that  the 
alkali  is  partly  saturated  and  the  power  of  the  blood  to  carry  CO.,  is  lessened, 
with  serious  results  (see  "Acid  Intoxication,"  Chap,  xx);  and,  second,  the  bacteri- 
cidal power  of  the  blood  is  found  to  vary  according  to  its  alkalinity.  In  fact, 
metabolic  activity  seems  generally  to  be  favored  V\v  certain  degrees  of  alkalinity; 
for  example,  J.  Loeb^-  found  that  sea-urchin  eggs  develop  with  much  greater 
rapidity  if  a  small  amount  of  OH  ions  is  free  in  the  .-:ea-water.  Brandenburg'^ 
states  that  the  non-diffusible  alkali  varies  according  to  the  amount  of  protein  in 
the  blood;  in  pneumonia  and  acute  nephritis  he  found  it  low.  In  cancer  the 
titrable  alkalinitj'  is  distinctly  increased,  and  Moore  and  Walker'*  hold  that  this  is 
due  to  an  increased  alkalinity  of  the  proteins  of  the  blood.  Awerbach'*  claims  that 
in  severe  high  fevers  the  bactericidal  effect  of  the  blood  alkalinity  is  increased 
(see  also  "Passive  Congestion"  for  further  discussion  concerning  the  relation  of 
alkalinity  to  bactericidal  power). 

Viscosity  of  the  Blood." — Normal  blood  is  about  five  times  (4.5  times,  Austrian) 
more  viscous  than  water,  chiefly  because  of  the  corpuscles  and  the  dissolved  pro- 
teins. This  viscosity  does  not  vary  directly  with  the  specific  gravity  or  the  hemo- 
globin, but  is  closely  related  to  the  number  of  red  corpuscles  (Burton-Opitz) ; 
laking  the  corpuscles  increases  the  viscosity  considerably.  Most  salts  increase 
the  viscositj',  but  some,  especially  iodides,  are  said  to  reduce  it.  Carbon  dioxide 
increases  viscosity  greatly,  even  when  in  amounts  possible  in  the  circulating  blood. 
Anemia  decreases  the  viscosity,  approximately  in  proportion  to  the  number  of 
corpuscles;  polj'cythemia  is  accompanied  by  a  corresponding  increase;  leukemia, 
because  of  anemia,  shows  a  decrease;  in  nephritis  there  maj'  either  be  an  increase 
or  a  decrease  in  the  viscosity,  not  corresponding  in  any  way  to  the  blood  pressure. 
Cardiac  disease  with  edema  shows  low  viscosity  because  of  the  anemia  and  hydre- 
mia, but  if  there  is  polycj'themia  and  no  edema  the  viscosity  may  be  high.  .Jaun- 
dice causes  an  increase,  diabetes  gives  variable  results.  Typhoid  causes  no  charac- 
teristic change  beyond  that  resulting  from  anemia,  and  in  pneumonia  the  cyanosis 
and  salt  retention  usually  cause  an  increase  (Austrian).  Gullbring'"  found  the 
viscosity  to  vary  directly  with  the  per  cent,  of  neutrophiles.  As  blood  viscosity 
depends  largely  upon  the  corpuscles,  it  increases  with  reduction  in  the  size  of  the 
lumen  of  the  tube  through  which  it  passes,  unlike  a  true  solution;  hence  with  narrow 
capillaries  the  viscosity  is  abnormally  high  until  we  reach  the  point  where  the 
corpuscles  plug  the  capillary. 

HEMORRHAGE 

Hemorrhages  result  from  an  altered  condition  in  the  vessel-walls, 
which  may  be  due  either  to  trauma  or  to  chemical  injuries.  Of 
the  chemical  agencies  causing  hemorrhages,  bacterial  products  arc  the 

11  See  Robertson,  Jour.  Biol.  Chem.,  1909  (6;,  313;  1910  (7),  351. 

1=  Arch.  f.  Entwicklungsmechanik,  1898  (7),  631. 

13  Deut.  med.  Woch.,  1902  (28),  78;  Zeit.  f.  klin.  Med.,  1902  (-45),  157. 

1^  Biochem.  Jour.,  1906  (1),  297;  good  discus.sion  of  blood  reaction. 

15  Med.  Obosrenije,  1903,  p.  596. 

1^  Review  of  literature  by  Determann,  Zeit.  klin.  Med.,  1910  (70),  185;  also 
Austrian,  Johns  Hopkins  Hosp.  Bull.,  1911  (22),  9.  See  also  Traube,  Internat. 
Zeit.  physik-chem.  Biol.,  1914  (1),  389. 

'"  Beitr.  klin.  Tuberk.,  1914  (30),  1. 


294  DISTURBANCES  OF  CIRCULATION 

most  important  practically,  but  many  poisons,  such  as  phosphorus, 
formaldehyde,  yhytotoxins  (ricin,  abrin,  and  crotin),  and  zootoxms 
(snake  venoms)  cause  numerous  and  abundant  hemorrhages.  For- 
merly, the  tendency  was  to  ascribe  hemorrhages  from  the  above  causes 
to  mechanical  injury  of  the  vessels  by  thrombi,  or  by  emboli  of  ag- 
glutinated corpuscles,  but  the  work  of  Flexner^^  has  shown  that 
venoms  cause  hemorrhages  by  injuring  the  capillary  walls,  so  that 
actual  rents  are  produced  by  the  intravascular  pressure,  and  it  seems 
highly  probable  that  hemorrhages  are  produced  by  other  chemical 
substances  in  a  similar  way.  We  may,  therefore,  refer  such  hemor- 
rhages to  an  endotheliotoxic  action  of  the  poison,  or  to  a  solvent  effect 
upon  the  intercellular  cement  substance.  In  the  case  of  ordinary 
chemical  poisons  the  endotheliotoxic  action  is  not  specific,  but  with 
some  of  the  toxins  it  seems  to  be  quite  so;  for  example,  rattlesnake 
venom  contains  an  endotheliotoxic  substance  ijiemorrhagin) ,  which 
seems  to  be  a  specific  poison  for  endothelium,  and  which  is  the  most 
dangerous  constituent  of  the  venom.  If  we  immunize  animals  against 
tissues  containing  much  endothelium  (e.  g.,  lymph-glands),  their  serum 
will  be  found  to  contain  endotheliotoxins,  so  that  when  tliis  serum 
is  injected  subcutaneously  into  a  susceptible  animal,  large  local  hemor- 
rhages result;  if  injected  into  the  peritoneal  cavity,  there  results 
marked  desquamation  of  the  endothelial  cells,  which  soon  undergo  de- 
generative changes  (Ricketts).^^  It  is  quite  probable  that  the  bac- 
terial poisons  that  cause  marked  hemorrhagic  manifestations  likewise 
contain  endotheliotoxins,  although  this  matter  does  not  seem  to  have 
been  investigated. 

Even  hemorrhage  by  diapedesis  seems  to  be  due  to,  or  at  least 
associated  with,  chemical  changes  in  the  capillary  walls,  for  Arnold-*' 
found  that  when  capillaries  from  which  diapedesis  had  occurred 
were  stained  by  silver  nitrate,  dark  areas  were  found  between  the 
endothelial  cells.  As  silver  nitrate  is  a  stain  for  chlorides,  and  dark- 
ens intercellular  substance  because  it  is  rich  in  sodium  chloride 
(Macallum),  it  is  probable  that  there  is  an  increase  in  the  amount 
or  a  difference  in  the  method  of  combination  of  the  chlorides  of  the 
cement  substance  between  the  endothelial  cells  at  the  places  where 
red  corpuscles  escape.  M.  H.  Fischer-^  suggests  that  diapedesis 
results  from  a  change  in  the  endothelial  cells,  which  under  the  inthi- 
ence  of  acids  or  other  agents  of  metabolic  origin  become  excessively 
hydrophihc,  swell  up,  and  become  so  softened  that  corpuscles  may 
pass  directly  through  the  cell,  just  as  a  drop  of  mercury  can  pass 
through  a  sufficiently  soft  jelly  without  leaving  a  hole  in  the  jelly. 

Ilemori'hage  in  cachetic  conditions  is  often  ascribed  to  changes 
in  the  vessel-walls  due  to  malnutrition,  but  it  is  diffi.cult  to  imagine 

"  Univ.  of  Penn.  Med.  Bull.,  1902  (15),  355. 
"  Trans.  Chicago  Path.  Soc,  1902  (5),  181. 
20  Viichow's  Arch.,  1875  (()2),  157. 
2'  "Nephritis,"  New  York,  1912,  p.  78. 


HEMOinaiAdE  295 

capillary  walls  suffering  from  lack  of  nourisliiiicnt,  even  with  the 
poorest  of  blood,  and  it  seems  more  probable  that  the  hemorrhages  are 
due,  even  in  cachexia,  to  chemical  constituents  of  the  blood  that  in- 
jure the  endothelium.  Hemorrhages  that  follow  re-establishment  of 
the  circulation  after  complete  occlusion,  however,  may  be  the  result 
of  asphyxial  clianges  in  the  capillary  walls,  presumably  colloidal  swell- 
ing of  the  cells. 

After  severe  hemorrhages  the  blood  shows  a  decrease  in  specific 
gravity  and  viscosity,  an  increase  in  surface  tension  and  electrical 
resistance,  and  either  increase  or  decrease  of  the  freezing-point  de- 
pression, all  these  changes  being  transient  if  the  individual  is  other- 
wise normal.'^  (See  also  Secondary  Anemia.)  There  is  a  rapid 
absorption  of  fluid  from  the  tissues  and  tissue  spaces,  resulting  in  a 
dilution  of  protein  and  formed  elements,  but  not  of  salts.  For  the 
same  reason  the  density  of  the  blood  decreases  in  direct  relation  to  the 
proportion  of  the  total  blood  that  has  been  lost.-^  The  alkali  reserve 
of  the  blood  is  somewhat  lowered  by  severe  hemorrhage, ^^  but  there  is 
not  a  marked  acidosis.  The  total  nitrogen  of  the  blood  of  course 
falls,  but  there  is  a  tendency  for  sugar,  urea  and  non-protein  N  to 
increase,  and  there  is  increased  elimination  of  creatine  in  the  urine, 
presumably  from  destruction  of  muscle  tissue  to  replace  the  lost  blood 
proteins.  There  is  said  to  be  a  decreased  permeability  of  vessels, 
resulting  in  reduced  exudative  processes. ^^  The  proportion  of  the 
several  blood  proteins  is  variably  altered  after  repeated  hemorrhages; 
the  sugar  is  little  affected^^  but  there  may  occur  a  marked  rise  in  the 
content  of  immune  bodies,  especially  specific  agglutinins.^^  Rapid 
hemorrhages  cause  a  decrease  in  the  coagulation  time  because  of  a 
decrease  in  antithrombin  and  a  slight  increase  in  prothrombin,  in 
spite  of  a  decrease  in  fibrinogen.-^  If  the  blood  is  withdrawn  re- 
peatedly in  large  amounts,  centrifuged,  and  the  washed  corpuscles 
reinjected  suspended  in  isotonic  salt  solution  (plasmaphaeresis),  life 
can  be  maintained  even  after  4  to  5  times  the  total  volume  of  blood 
has  been  removed  and  washed.  This  is  possible  because  of  rapid 
reformation  of  the  plasma,  and  the  blood  shows  the  changes  characteris- 
tic of  secondary  anemias. ^^  Lipemia  is  often  produced  by  severe  or 
repeated  hemorrhages,  with  a  great  increase  in  the  phospholipins  of 
the  plasma  and  corpuscles. ^^^ 

Changes  in  the  Extravasated  Blood. — These  begin  soon  after 
its  escape.     In  most  situations  sufficient  fibrin  ferment  is  formed  to 

"  Oliva,  Folia  clinica,  1912  (3),  213. 

"Richet  et  al,  Compt.  Rend.  Acad.  Sci.,  1918  (166),  587. 

2<Buell,  Jour.  Biol.  Chem.,  1919  (40),  29;  Tatum,  ibid.,  1920  (-41),  59. 

"Luithlen,  Med.  Klin.,  1913  (9),  1713. 

26  Taylor  and  Lewis,  Jour.  Biol.  Chem.,  1915  (22),  71. 

''^  See  Hahn  and  Langer,  Zeit  Immunitat,  1917  (26),  199. 

28  Drinker,  Ainer.  Jour.  Phvsiol.,  1915  (36),  305. 

29  Abel  et  al,  Jour.  Pharmacol.,  1914  (5),  625;  1915  (7),  129. 
29"Bloor  and  Farrington,  Jour.  Biol.  Chem.,  1920  (41)  xlviii. 


296  DISTURBANCES  OF  CIRCULATION 

cause  prompt  clotting,  but  in  the  pleura  and  other  serous  cavities  the 
blood  may  remain  fluid  for  some  time,  possibly  because  of  lack  of 
cellular  injury  that  might  cause  liberation  of  fibrin  ferment. ^°  If  the 
blood  does  not  become  infected,  the  rapidity  of  subsequent  changes 
depends  chiefly  upon  the  location  and  amount  of  blood.  Small  ex- 
travasations of  blood  into  the  tissues  are  subjected  to  the  action  of  the 
tissue  cells  and  of  leucocytes  emigrating  freely  from  the  capillaries; 
large  masses  of  blood  are  but  little  affected  by  these  agencies,  the 
leucocytes  within  the  mass  soon  die,  and  secondary  changes  go  on  very 
slowly.  In  small  subcutaneous  hemorrhages  (e.  g.,  a  bruise)  enzymes 
from  the  invading  leucocj^tes  and  tissue-cells  soon  dissolve  the  small 
quantities  of  fibrin  present;  even  earlier  the  stroma  of  the  red  cor- 
puscles is  so  altered  that  hemolysis  occurs  and  the  hemoglobin  escapes 
and  diffuses  into  the  tissues.  This  hemolysis  may  be  brought  about 
by  the  action  of  proteolytic  enzymes  on  the  corpuscles,  or  bj^  the  hemo- 
lytic action  of  the  products  of  protein  splitting.  Soon  the  hemoglo- 
bin disintegrates,  forming  the  masses  of  pigment  so  characteristic 
of  old  hemorrhagic  areas,  and  also  giving  rise  to  the  discoloration 
observed  beneath  the  skin  in  the  later  stages  of  resorption  of  hemor- 
rhagic extravasations.  The  first  products  of  the  splitting  of  hemo- 
globin are:  (1)  The  protein,  glohin,  which  constitutes  94  per  cent, 
of  the  hemoglobin;  and  (2)  the  iron-containing  coloring-matter,  hem- 
atin  (in  the  absence  of  oxygen  the  pigment  is  reduced  hematin  or 
hemochromogen) .  As  hematin  may  be  experimentally  obtained  by 
the  action  of  proteases  upon  hemoglobin,  the  decomposition  of  the 
hemoglobin  in  the  tissues  is  probably  accomplished  in  a  similar  way 
by  the  proteases  of  the  leucocj'^tes,  tissue-cells  and  blood  plasma;  the 
globin  is  thus  digested  away  and  the  soluble  products  carried  off, 
while  the  insoluble  hematin  remains. ^^  The  hematin  gradually  un- 
dergoes further  changes,  forming  an  iron-free  pigment  (hematoidw) 
and  an  iron-containing  pigment  [hemosiderin) . 

Hematoidin  is  nearly  or  quite  identical  with  the  bile-pigment,  hili- 
rubin,  and  is  absorbed  from  the  hemorrhagic  extravasation  and  elimi- 
nated as  bilirubin  in  the  bile.  Possibly  some  of  the  hematoidin 
undergoes  transformation  into  urobilin,  and  is  then  eliminated  in  the 
urine.  Hemosiderin  seems  to  be  relatively  insoluble  and,  therefore, 
is  more  slowly  removed,  so  that  it  may  be  found  at  the  site  of  a  hem- 
orrhage after  the  other  evidences  of  blood  extravasation  have  been 
removed.  It  may  be  easily  demonstrated  by  staining  with  potassium 
ferrocyanide,  the  Prussian  blue  that  is  formed  being  readily  dis- 
tinguished.    Unstained  hemosiderin  generally  appears  in  the  form 

3"  Denny  and  Minot  (Anier.  Jour.  Physiol.,  1916  (30),  455)  believe  that  the 
blood  really  does  clot,  and  that  it  remains  lluid  wiuMi  withdrawn  because  the 
fibrinogen  has  l)een  removed  by  clotting.  Zahn  and  Walker  (Biochem.  Zeit.,  1913 
(58)^  130),  however,  consider  that  the  fibrinogen  is  altered  by  the  pleural  endo- 
thelium, so  that  it  cannot  clot. 

^'  More  fully  discussed  in  the  consideration  of  "Pigmentation,"  (^hap.  xviii. 


llEMOUiniACK  297 

of  brown  or  yellowish-brown  granules,  never  as  crystals.  After  a 
time  the  hemosiderin  is  taken  away,  and  probably  is  to  a  greater  or 
less  extent  deposited  in  the  liver  and  spleen,  either  as  hemosiderin 
or  as  some  other  iron  compound.  Eventually  it  is  probably  utilized 
to  make  new  hemoglobin;  at  any  rate,  the  iron  liberated  by  the  break- 
ing up  of  hematin  within  the  body  does  not  appear  to  be  eliminated.'^ 

The  changes  in  the  red  corpuscles  described  above  are  not  at  all 
peculiar  to  cxtravasated  blood,  but  are  quite  the  same  as  the  changes 
that  are  going  on  continuously  and  normally  in  the  blood.  Red  cor- 
puscles are  short-lived,  being  but  non-nuclcatcd  fragments  of  cells, 
and  they  are  continually  disintegrating  with  the  production  of  iron- 
free  pigments  that  are  excreted  as  the  coloring-matters  of  the  bile  and 
urine,  while  the  iron  is  worked  over  again  into  new  hemoglobin  after 
a  varying  period  of  storage  in  the  tissues,  particularly  in  the  spleen 
and  liver.  The  destruction  of  red  corpuscles  under  normal  conditions 
seems  to  take  place  chiefly  in  the  spleen,  bone-marrow,  and  hemo- 
lymph  glands,  where  injured  or  decrepit  corpuscles  are  taken  out  of 
the  blood  by  the  phagocytic  endothelial  cells,  and  decomposed  by 
intracellular  enzymes.  In  hemorrhagic  extravasations  the  changes 
are  essentially  the  same;  some  corpuscles  are  destroyed  by  phago- 
cytes, but  more  by  extracellular  enzymes.  The  products  of  decom- 
position also  seem  to  be  no  difTerent  from  those  formed  during 
normal  katabolism  of  hemoglobin,  and  they  meet  the  same  fate  in 
the  end. 

If  the  hemorrhages  are  very  abundant,  some  hemoglobin  may  be 
absorbed  as  such  and  appear  in  the  urine,  but  this  probably  seldom 
happens  unless  red  corpuscles  are  also  being  destroj-ed  in  the  circu- 
lating blood. '^  An  increased  amount  of  iron  accumulates  in  the 
liver,  but  if  much  blood  has  been  lost  by  hemorrhage  on  free  surfaces, 
the  iron  content  of  the  liver  is  decreased,  as  it  is  taken  away  to  form 
new  hemoglobin  (Quincke).'*  Excretion  of  bile-pigments  is  increased 
by  destruction  of  blood  (Stadelmann),  but  not  greatlj^  in  the  case  of 
internal  hemorrhages,  for  the  blood  is  decomposed  and  absorbed 
too  slowly.  Schurig'^  found  that  hemoglobin  injected  into  the  tissues 
is  partly  decomposed  in  situ  with  formation  of  iron  compounds, 
but  the  greater  part  enters  the  circulation  as  hemoglobin,  and  is 
partly  converted  into  bile-pigment  by  the  liver-cells,  the  rest  being 
converted  into  simpler  iron  compounds  by  the  spleen,  bone-marrowy, 
and  renal  cortex. 

If  the  hemorrhagic  extravasation  has  been  large  in  amount,  the 
deeper  portions  of  the  mass  are  not  soon,  if  ever,  invaded  by  leucocytes 

32  See  Morishima,  Arch.  f.  exp.  Path.,  1898  (41),  291. 

'3  In  cerebral  hemorrhage  the  blood  serum  may  be  greenish  and  somewhat 
fluorescent  from  absorbed  pigment,  according  to  Marie  and  Leri,  Union  Pharra., 
Aug.  15,  1914. 

"  Deut.  Arch.  klin.  Med.,  1880  (25).  567;  1880  (27),  193. 

36  Arch.  exp.  Path.  u.  Pharm.,  1898  (41),  29. 


298  DISTURBANCES  OF  CIRCULATION 

or  tissue-cells.  Consequently  the  blood  is  acted  upon  very  slowly 
by  the  enzymes  liberated  by  the  leucocytes  it  contains  itself,  and  by 
the  small  amounts  of  proteases  in  the  serum.  Furthermore,  the  prod- 
ucts of  decomposition  are  not  soon  absorbed,  but  accumulate  in  con- 
siderable amounts,  so  that  we  often  find  crystalline  deposits  of 
hematoidin,  sometimes  even  of  hematin,  hemoglobin,  or  parahenioglobin 
(Nencki)^^  or  methemoglohin. 

The  least  soluble  constituent  of  the  red  corpuscle  stroma,  choles- 
terol, also  accumulates  in  such  extravasations  as  large,  thin  plates; 
after  most  of  the  other  products  of  disintegration  have  been  absorbed 
from  such  accumulations  of  blood,  the  most  conspicuous  part  of  the 
residue  may  be  a  mass  of  cholesterol  crystals  imbedded  in  prolifer- 
ating connective  tissue. 

Hemophilia" 

There  are  several  pathological  conditions  associated  with  increased 
tendency  to  bleeding,  notably  scurvy  and  the  various  forms  of  pur- 
puras, but  especially  the  remarkable  hereditary  condition,  hemophilia. 
In  the  purpuric  diseases  various  of  the  factors  concerned  in  coagula- 
tion of  the  blood  have  been  found  altered, ^^  notably  the  blood  plate- 
lets,^^ but  Howell  found  no  change  in  either  prothrombin  or  anti- 
thrombin  in  yurpura  hemorrhagica  and  other  related  conditions.  Simi- 
lar negative  results  were  obtained  in  scurvy  by  Hess.^°  Melena 
neonatorum  exhibits  decreased  prothrombin  in  the  blood,  while  in 
leukemias  and  anemias  there  may.be  an  excess  of  antithrombin,^' 
leading  to  severe  hemorrhage  (see  also  Thrombosis). 

Since  hemophilia  seems,  superficially  at  least,  to  depend  upon  some 
alteration  in  a  chemical  property  of  the  blood,  namely,  coagulability, 
it  is  frequently  regarded  as  an  example  of  hereditary  transmission 
of  a  chemical  abnormality.  The  exact  cause  of  this  peculiar  tendency 
to  prolonged  bleeding  from  insignificant  or  perhaps  imperceptible 
wounds  has  been  sought  vigorously  by  both  histological  and  chemical 
means,  but  as  yet  without  avail.  Various  observers  have  described 
abnormal  thinness,  or  increased  cellularity  or  fatty  degeneration  of 
the  vessel-walls,  but  the  findings  have  been  far  too  inconstant  to 
afford  a  satisfactory  anatomical  explanation  of  all  the  features  of 
hemophilia.     Likewise  increased  blood  pressure  can  be  ruled  out,  for 

3"  Arch.  exp.  Path.  u.  Pharni.,  1S8G  (20),  332.^ 

"  Ijiterature  and  r6suin6  given  bv  Stenipcl,  Cent.  f.  Grcn7,p;eb.  Med.  u.  Chir., 
1900  (;i),  75;i;  Sahli,  Zeit.  f.  klin.  Med.,  1<)05  (5(>),  29-4;  Marcliand,  in  Krehl  and 
Marchand's  Handb.  allg.  Pathol.,  1912,  II  (1),  307.  Also  later  references  in  this 
text. 

^^  See  Ilurwitz  and  Lucas,  Arch.  Int.  Med.,  191G  (17),  543;  Minot  et  al,  ibid., 
191(5  (17),  101. 

'"See  Lee  and  Robertson,  Jour.  Med.  Res.,  1916  (3;j),  323;  Hess,  Proc.  See. 
Exp.  Biol.  Med.,  1917  (14),  96. 

^0  Ainer.  .Jour.  Dis.  Children,  1914  (8),  386. 

"  Whipple,  Arch.  Int.  Med.,  1913  (12),  037. 


HEMOPHILIA  299 

although  the  left  heart  is  frequently  enhirf^ed,  there  is  usually  no  in- 
creased blood  pressure  demonstrable;  furthermore,  conditions  of 
high  blood  pressure,  such  as  nephritis,  do  not  cause  hemophilia.  The 
theory  of  ''hydremic  plethora"  is  also  without  good  foundation. 

The  most  natural  place  to  look  for  the  fundamental  fault  is  in  the 
blood,  but  speaking  strongly  against  this  is  the  occasional  occurrence 
of  "local"  hemophilia;  e.  g.,  in  this  type  of  hemophilia  wounds  of  the 
skin  may  behave  as  in  normal  individuals,  whereas  any  injury  of  the 
mucous  surfaces  is  followed  by  pronounced  hemophilic  bleeding ;^^ 
in  other  cases  the  hemophilic  bleeding  is  limited  to  regions  above  the 
shoulders;  in  still  another  class  the  bleeding  is  always  from  one  organ, 
e.  g.,  the  kidneys.  Nevertheless,  a  great  deal  of  investigation  of  the 
blood  has  been  done,  at  first  chiefl}'  with  negative  results.  There  are  no 
characteristic  changes  in  the  cellular  elements  of  the  blood,  beyond 
the  changes  common  to  all  secondary  anemias,  excepting  possibly  a 
decrease  in  the  number  of  w^hite  corpuscles  with  a  relative  increase  in 
the  number  of  lymphocytes  as  observed  by  Sahh;  the  platelet  count 
is  normal.  No  constant  alterations  in  the  salts  of  the  blood  have 
been  found,  calcium  usually  being  normal  ;^^  and  the  proportion  of 
water,  fibrinogen  and  the  several  other  proteins,  the  alkalinity,  and 
the  osmotic  pressure  of  the  serum  all  seem  to  be  normal.  Metabo- 
Hsm  is  unchanged,  except  possiblj^  for  calcium  loss  in  some  cases.** 
Since  bleeding  is  normally  stopped  principally  by  coagulation,  a  de- 
ficiency in  fibrin  or  its  antecedents  might  be  expected,  but  most 
studies  on  this  point  have  shown  a  normal  amount  of  fibrinogen  in 
the  blood  of  hemophilics,  the  frequent  formation  of  large  tumors  of 
clotted  blood  at  the  bleeding  points  supporting  the  experimental 
evidence  that  the  blood  contains  an  abundance  of  fibrinogen.  The 
"bleeding  time"  following  punctures  in  the  skin  is  not  excessive.  As 
to  the  rate  of  clotting,  Sahli,"  who  avoided  a  number  of  errors  made 
in  earlier  investigations,  found  that  in  the  intervals  between  the  at- 
tacks of  hemorrhage  the  rate  of  the  coagulation  of  the  blood  is  con- 
stantly much  slower  than  normal.  During  an  attack  of  bleecUng  the 
coagulation  time  approaches  the  normal;  indeed,  it  may  be  faster 
than  normal;  apparently  this  is  due  to  a  reaction  on  the  part  of  the 
organism  to  the  loss  of  blood.  If  blood  is  collected  directly  from  the 
site  of  bleeding  the  coagulation  time  is  very  rapid,  because  of  the  ac- 
cumulation of  fibrin  ferment  from  the  clot  over  which  the  escaping 
blood  flow^s.  Yet  in  spite  of  the  normal  coagulability  of  the  blood  and 
the  rapid  clotting  after  the  blood  escapes  from  the  vessel,  bleeding 
continues  for  long  periods  before  it  can  be  stopped.  As  he  found  no 
general  change  in  the  properties  of  the  blood  to  account  for  the  bleed- 

"  Abderhalden,  Ziegler's  Beitr.,  1904  (35),  213. 
"  Ivlinger  and  Berg,  Zeit.  klin.  Med.,  1918  (85),  335,  406. 
"  Kahn,  Amer.  Jour.  Dis.  Children,  1916  (11),  103;  Laws  and  Cowie,  ibid. 
1917  (13),  236;  Hess,  Bull.  Johns  Hopkins  Hosp.,  1916  (26),  372. 


300  DISTURBANCES  OF  CIRCULATION 

ing,  and  as  local  influences  seem  to  be  important  in  hemophilia,  Sahli 
advanced  the  plausible  hypothesis  that  the  cause  of  hemophilia  lies 
in  hereditary  deficiency  of  the  fibrin-forming  substances,  thi'om- 
bokinase  or  zymoplastic  substance  (see  "Thrombosis"),  in  the  vessel- 
walls,  so  that  when  the  vessels  are  injured  there  is  no  local  production 
of  fibrin  such  as  occurs  normally.  Local  hemophilia  may  be  explained 
readily  as  a  local  deficiency  in  fibrinoplastic  material.  In  general 
hemophilia  even  the  leucocytes  may  exhibit  the  same  defect,  in  which 
case  clotting  of  the  blood  is  diminished  even  outside  the  tissues.  This 
hypothesis  seems  to  be  in  excellent  agreement  with  many  of  the  facts 
now  known,  but  there  yet  remains  to  be  demonstrated  such  a  lack  ol" 
fibrin-forming  elements  in  the  vessel-walls  and  other  tissues  of  a  hemo- 
philic subject,  and  a  single  autopsy  of  a  hemolytic  subject  gave,  on  the 
contrary,  a  very  active  thromboplastic  extract  from  the  vessels 
(Gressot).^^  The  tissues  of  one  case  studied  by  Lowenburg  and 
Rubenstone,*^  however,  showed  in  the  liver  and  thyroid  a  decreased 
capacity  to  accelerate  coagulation. 

With  the  improved  methods  of  study  of  the  factors  in  coagulation 
of  blood  introduced  by  Howell,  it  has  been  found  by  him  and  cor- 
roborated by  others^'^  that  in  hemopliilia  there  is  constantly  a  defi- 
ciency in  prothrombin,  the  other  factors  being  practically  normal  in 
amount,  and  as  in  other  hemorrhagic  conditions  there  is  no  equal 
alteration  in  the  prothrombin,  they  look  upon  this  change  as  an 
essential  characteristic  of  hemopliilia.  Fonio,  and  jXIinot  and  Lee, 
however,  find  that  the  blood  platelets  of  hemophihcs  are  remarkably 
ineffective  in  causing  coagulation  of  either  normal  or  hemophilic 
plasma,  although  normal  platelets  cause  normal  coagulation  of  hemo- 
philic plasma,  and  therefore  conclude  that  there  is  some  deficient 
activity  on  the  part  of  the  platelets  in  spite  of  their  occurrence  in  nor- 
mal numbers  in  hemopliilia.  The  significance  of  the  platelets  is  shown 
especially  clearly  by  the  observation  of  Ledingham  and  Bedson*^ 
that  antiplatelet  serum  will  produce  a  purpuric  condition  when  in- 
jected into  animals  of  the  species  furnishing  the  platelets,  although 
no  similar  effect  is  produced  by  antileucocj'te  or  antierythrocyto 
serum,  Hess^^  states  that  there  may  be  an  hereditary  purjnira, 
sometimes  occurring  in  the  females  of  hemophilic  families,  difl'ering 
from  hemophilia  in  a  deficiency  in  the  number  of  platelets,  hemor- 
rhages following  local  congestions  or  puncture  wounds  and  exhibiting 
an  increase  in  the  "bleeding  time." 

"Zeit.  klin.  Med.,  1912  (76),  194.     Since  corroborated  bv  Minot  and  Lee." 

"Jour.  A.mer.  Med.  Assoc,  1918  (71),  1196. 

*'  HowoU,  .Vroh.  Int.  Med.;  1914  (VA),  Tti;  llurwitz  and  Lucas,  ibid.,  1916  (17), 
543;  Minot  and  Lee,  ibi<l.,  lUKi  (18),  474;  Klingcr,  Zeit.  klin.  Med.,  1918  (86) 
335;  Pcttibonc,  Jour.  Lab.  Clin.  Sled.,  1918  (3),  275;  these  papers  review  recent 
work  on  hemophilia. 

■•*  Lancet,  Feb.  13,  1915.  Similar  observations  have  been  made  by  Watabiki 
(Kitasato's  Arch.  Exp.  Med.,  1917  (1),  195). 

"Arch.  Int.  Med.,  1916  (17),  203. 


ANEMIA  AND  THE  SPECIFIC  ANEMIAS  301 

ANEMIA  AND  THE  SPECIFIC  ANEMIAS" 

The  customary  although  unsatisfactory  and  unscientific  division 
of  the  anemias,  is  into — ■  (a)  'primary,  i.  e.,  those  in  which  the  anemia 
seems  to  depend  upon  some  abnormahty  in  the  blood-forming  organs 
or  in  the  blood  itself;  and  (6)  secondare/,  embracing  anemias  the  result 
of  some  obvious  cause,  such  as  hemorrhage,  poisoning  with  blood- 
destroying  poisons,  cachexia,  etc.  In  these  various  forms  of  anemia 
certain  chemical  differences  prevail,  but  they  are  by  no  means  so  strik- 
ing as  arje  the  histological  differences  in  the  formed  elements  of  the 
blood. ^1 

Secondary  Anemias 

As  the  simplest  variety,  anemia  following  a  single  large  hemorrhage 
may  be  considered  first. 

If  loss  of  blood  by  hemorrhage  is  rapid,  the  effects  are  naturally 
much  more  serious  than  when  the  loss  is  slow.  The  total  quantity  of 
blood  in  the  average  adult  is  estimated  at  about  3d^3  to  ^5  the  total 
body  weight  (.therefore  about  10  to  12  pounds),  although  this  propor- 
tion does  not  hold  for  extremely  obese  or  extremely  thin  individuals; 
in  infants  the  proportion  is  lower — about  ^^o-  When  one-third  of 
the  total  amount  of  blood  is  lost  rapidly,  a  marked  fall  of  blood  pres- 
sure occurs;  loss  of  one-half  of  the  total  amount  may  be  fatal,  and  loss 
of|more  than  that  at  one  time  usually  is  fatal.  The  chief  cause  of 
death  following  large  hemorrhages  is  the  low  blood  pressure  rather 
than  the  loss  of  any  of  the  constituents  of  the  blodd;  hence  the  suc- 
cessful results  of  the  use  of  physiological  salt  solution  after  severe 
hemorrhage.  The  number  of  corpuscles  may  be  greatly  reduced  after 
several  small  hemorrhages,  even  to  as  low  as  11  per  cent,  of  the  normal 
number  (Hayem),  without  fatal  results,  because  in  the  intervals  be- 
tween the  hemorrhages  enough  fluid  has  been  taken  up  by  the  blood 
to^maintain  the  blood  pressure  within  safe  limits.  After  a  severe 
hemorrhage  the  composition  of  the  blood  changes  rapidly,  for  the 
fluids  contained  within  the  tissues  and  lymph-spaces  pass  into  the  blood 
in  large  amounts.  This  helps  to  maintain  blood  pressure,  but  results 
in  the  blood  containing  a  large  proportion  of  water  and  salts  and  a  smal- 
ler amount  of  protein  and  red  corpuscles;  the  "total  alkalinity"  also 
falls,  largely  because  of  the  scarcity  of  ''fixed  alkali, "  on  account  of  the 
poverty  in  corpuscles  and  blood  proteins.  The  proportion  of  water 
increases  at  first  more  rapidly  than  the  proportion  of  salts,  and  as  a 
consequence  the  size  of  the  red  corpuscles  is  increased  because  of  im- 
bibition of  water;  indeed,  it  is  possible  that  this  may  even  be  sufficient 
to  cause  hemolysis,  which  will  happen  if  the  isotonic  strength  of  the 

*"  Metabolism  in  anemia  reviewed  bj'  Mohr,  Handbuch  d.  Biochem.,  1910  (IV 
(2)),  372. 

^'  Concerning  local  anemia,  see  "Infarcts." 


302  DISTURBANCES  OF  CIRCULATION 

blood  becomes  less  than  that  of  a  0.46  per  cent.  NaCl  solution  (Lim- 
beck), while  swelling  may  occur  whenever  the  strength  is  below  0.8 
per  cent.  The  specific  gravity  of  the  erthrocytes  is  decreased;^^ 
the  depression  of  the  freezing  point  increases,^^  while  the  viscosity 
falls.     The  number  of  platelets  is  high. 

Regeneration  of  the  blood  begins  very  soon,  and  for  some  time  the 
number  of  corpuscles  exceeds  the  proportion  of  hemoglobin.  During 
this  time  the  amount  of  iron  in  the  liver  and  spleen  is  decreased,  it 
being  taken  up  to  be  used  in  the  formation  of  new  hemoglobin.  The 
rate  of  regeneration  is  much  increased  by  a  meat  diet,  but  not  by 
iron  administration.^^  Red  corpuscles  do  not  regenerate  in  animals 
kept  on  an  incomplete  protein  diet  (ghadin).  If  the  hemorrhages 
are  numerous  and  the  condition  of  anemia  prolonged,  secondary 
changes  in  the  viscera  may  occur,  fatty  metamorphosis  being  most 
marked,  supposedly  because  of  decreased  oxidation.  Indeed,  many 
observers  state  that  repeated  bleedings  greatly  increase  body  weight 
by  causing  increased  fat  deposition. 

Metabolic  Changes. — Gies*^  studied  the  metabolism  of  dogs  after  withdrawing 
a  total  amount  of  blood  equal  to  11.5  per  cent,  of  the  body  weight  during  four 
bleedings,  and  found  that  a  slight  and  temporary  increase  in  nitrogenous  elimina- 
tion followed  the  bleedings,  owing  to  an  increased  protein  katabolism.  Basal 
metabolism  may  also  be  increased  in  anemia.^''  Sugar  increases  in  the  blood, 
while  albumin  and  lactic  acid  appear  in  the  urine.  After  each  successive  hemor- 
rhage the  proportion  of  fibrin  and  the  coagulability  of  the  blood  increase,  while 
the  proportion  of  the  ash  obtained  from  both  blood  and  serum  remains  practically 
unchanged  (Meyer  and  Gies).  Baumann"  states  that  in  regeneration  after  hem- 
orrhage the  serum  albumins  increase  more  rapidly  than  the  globulins,  while  others 
have  observed  the  opposite  relation.  The  urine  in  secondary  anemia  shows  the 
effects  of  increased  protein  katabolism,  its  specific  gravity,  total  solids,  and  total 
nitrogen  being  raised;  the  total  amount  of  urine  is  at  first  diminished  because  of 
lowered  blood  pressure,  but  it  soon  rises  above  normal  and  later  falls  back  to 
normal.  The  view  formerly  held  that  oxidation  is  decreased  in  anemia  has  been 
considerably  modified  by  more  recent  investigations;"^  in  fact,  respiration  studies 
indicate  heightened  gas  exchange  in  secondary  anemia.^* 

Secondary  anemia  due  to  cachexia,  or  to  malnutrition,  is  ac- 
companied by  a  general  decrease  in  all  the  elements  of  the  blood,  both 
cellular  and  chemical.  The  proteins  of  the  plasma,  particularly,  show 
a  decrease  in  starvation,  being  drawn  on  by  the  cells  for  food,  and 
the  total  quantity  of  blood  as  well  as  of  each  of  its  constituents  is  de- 
creased (Panum),''''  but  the  proportion  of  blood  to  body  weight  re- 
mains about   normal.     With  protracted  starvation  there  is  only  a 

«  Bonninger,  Zeit.  exp.  Path.,  1912  (11),  1. 

"  Iloesslin,  Ilofnicister's  Beitr.,  1906  (S),  431. 

6^  Hooper  and  Whipple,  Amer.  Jour.  Physiol,  1918  (45),  573. 

"American  Med.,  1904  (8),  155  (r6sum  6  of  literature). 

68  Review  by  Tompkins  el  al.,  Arch.  Int.  Med.,  1919  (23),  441. 

"  Jour.  Physiol.,  1903  (29),  18. 

68  See  Mohr,  Zeit.  exp.  Path.,  1906  (2),  435. 

sfGrafe,  Dent.  Arch.  klin.  Med.,  1915  (118),  148. 

90  Virchow's  Arch.,  1864  (29),  241. 


CHLOROSIS  303 

slight  loss  of  hemoglobin  and  an  increased  coagulability,  but  practi- 
cally no  other  changes/''  In  aplastic  anemias  the  prothrombin  and 
platelet  content  are  likely  to  be  low,  with  normal  fibrinogen/^ 

Anemia  due  to  hemolytic  agencies  presents  quite  different  fea- 
tures, in  that  red  corpuscles  are  almost  solely  attacked  and  the  prod- 
ucts of  their  disintegration  are  present  in  the  plasma.  As  a  result, 
the  plasma  or  serum  may  contain  free  hemoglobin,  and  if  the  hemo- 
globin is  in  large  amounts,  it  may  escape  into  the  urine.  Thus  par- 
oxysmal hemoglobinuria  is  probably  due  to  the  presence  in  the  blood  of 
hemolytic  substances,  which  can  be  demonstrated  in  the  blood  of  the 
patients  during  the  attack.  (See  Chapter  ix.)  The  products  of  the 
decomposition  of  the  hemoglobin  set  free  by  hemolysis  are  present  not 
only  in  the  blood,  but  also  in  the  organs,  particularly  the  liver  and 
spleen,  which  become  rich  in  iron.  In  acute  anemia  produced  by 
hemolytic  sera,  with  destruction  of  more  than  half  the  blood  in  three 
days,  nearly  all  the  iron  from  the  destroj^ed  hemoglobin  can  be  found 
in  the  liver,  spleen  and  kidneys,  there  being  but  little  lost  through  the 
urine  even  in  so  severe  an  anemia  as  this  (IMuir  and  Dunn).^^  Excre- 
tion of  bile-pigments  increases,  and  ^^hematogenous  jaundice"  may 
result,  the  bile-pigments  that  are  present  in  the  blood  being  derived 
from  the  hematoidin  of  the  hemoglobin  molecule.  Changes  in  metab- 
olism occur  which  are  quite  similar  to  those  observed  in  other  forms 
of  anemia,  with  fatty  changes  in  all  the  parenchymatous  organs,  in- 
creased protein  katabolism,  and  an  excessive  quantity  of  pigmentary 
substances,  particularly  urobilin,  in  the  urine.  The  plasma  chlorides 
are  increased. ^^  i 


[Chlorosis 

The  characteristic  feature  of  the  blood  in  chlorosis  is  the  rela- 
tively small  amount  of  hemoglobin  in  proportion  to  the  number  of 
corpuscles.  Apparently,  therefore,  the  fault  lies  rather  in  the  manu- 
facture of  hemoglobin  than  in  either  a  destruction  or  a  deficient  forma- 
tion of  red  corpuscles.  Erben's^^  analyses  of  chlorotic  blood  showed 
that  the  total  amount  of  protein  is  decreased,  chiefly  because  of  the 
deficiency  of  hemoglobin;  the  relation  of  serum  globulins  and  serum 
•albumin  is  unchanged,  while  the  proportion  of  fibrinogen  is  increased. 
There  is  much  more  fatty  substance  than  normal  in  both  the  serum 
and  the  erythrocytes,  but  the  lecitliin  is  decreased  both  in  the  serum 

"  Ash,  Arch.  Int.  Med.,  1914  (14),  8. 

"  Drinker  and  Hurwitz,  Arch.  Int.  Med.,  1915  (15J,  733;  Jour.  Exp.  Med., 
1915  (21),  401. 

"  Jour.  Path,  and  Bact.,  1915  (19),  417.  See  also  Dubin  and  Pearce,  Jour.  Exp. 
Med.,  1918  (27),  479. 

"  Steinfield,  Arch.  Int.  Med..  1919  (23),  511. 

"  Zeit.  klin.  Med.,  1902  (47),  302.  See  also  Frohmaier,  Folia  Hematol.,  1915 
(20),  115;  Beumer  and  Burger,  Zeit.  exp.  Path.,  1913  (13\  351. 


304  DISTURBANCES  OF  CIRCULATION 

and  in  the  total  blood,  although  somewhat  increased  in  the  red  cells. 
Cholesterol  is  decreased  in  both  serum  and  corpuscles.  In  the  ash, 
phosphoric  acid,  potassium,  and  iron  are  decreased,  while  calcium  and 
magnesium  are  both  increased.  An  apparent  increase  in  socUum  chlo- 
ride exists,  but  it  is  only  apparent,  being  the  result  of  the  increase  in 
the  proportion  of  plasma  m  the  blood.  The  total  amount  of  plasma 
is  greatly  increased  (polyplasmia) . 

The  decrease  in  hemoglobin  is  demonstrable  chemically'  as  well  as 
microscopically,  Becquerel  and  Rodier^^  having  found  the  amount  of 
iron  in  the  total  blood  decreased  in  direct  proportion  to  the  apparent 
decrease  in  hemoglobin,  which  frequently  falls  to  30-40  per  cent.,  and 
may  drop  to  20  per  cent.,  or  possibly  less.  Alkalinity',  as  determined 
by  titration,  is  diminished  in  some  cases,  but  generally  remains  nearly 
normal.  The  corpuscles  are  said  to  contain  a  larger  proportion  of 
water  than  normal,  independent  of  the  proportion  of  water  present 
in  the  serum.  Limbeck  found  their  isotonicity  (J.  e.,  the  strength  of 
NaCl  necessary  to  prevent  hemolysis)  very  low — -about  0.38-0.4  per 
cent.  NaCl. 

Very  few  changes  seem  to  occur  in  the  organs  of  the  body;  the 
usual  tendency  to  lay  on  fat,  and  the  occurrence  of  fatty  degeneration 
observed  commonly  in  anemias,  may  be  exhibited,  and  are  correlated 
with  Erben's  observation  of  an  increased  fat  content  in  the  blood; 
but  these  changes  are  often  absent.  The  hypoplasia  of  the  aorta, 
upon  which  Virchow  laid  so  much  stress,  is  now  considered  to  be  of 
little  or  no  significance.  Thrombosis  is  a  not  infrequent  complication 
of  chlorosis,^^  and  is  probably  favored  by  the  increased  platelet  and 
fibrin-content  of  the  blood  and  the  tendency  to  fatty  changes  in  the 
vessel-walls. 

Studies  of  nitrogenous  metabolism  bj'  Vannini^^  showed  practically 
no  alterations  except  a  slight  retention  of  nitrogen. 

Etiology. — As  to  the  etiology  of  chlorosis,  chemical  findings  indi- 
cate some  possibilities  and  negative  others,  but  decide  nothing.  That 
chlorosis  does  not  depend  upon  a  hemolytic  poison  is  well  established 
by  the  following  facts:  there  is  no  free  hemoglobin  in  the  blood  plasma, 
and  even  less  iron  in  the  serum  ash  than  normal;  lecithin  and  choles- 
terol, important  products  of  disintegration  of  erythrocytes,  are  both 
decreased  in  the  serum;  hematogenous  icterus  does  not  occur,  and 
the  amount  of  pigments  in  the  urine  and  feces  is  decreased. 

Apparently,  therefore,  hematogenesis  is  at  fault,  particularly  the 
formation  of  hemoglobin,  since  this  is  more  deficient  than  is  the  total 

*'  For  literature  see  Krehl,  "Basis  of  Symptoms,"  191G,  j).  100;  Ewing,  "Clin- 
ical Pathology  of  the  Blood,"  1901.  p.  167;  Kossler,  Cent.  f.  inn.  Med.,  1897  (18), 
657. 

"  See  Schweitzer,  Virchow's  Arch.,  1898  (152),  337,  and  Leichtonstern,  Miinch. 
med.  Woch.,  1899  (46),  1603. 

•i*  Virchow's  Arch.,  1904  (176),  375. 


PERNICIOUS  ANEMIA  305 

number  of  red  corpuscles.  The  r;ii)id  improvement  in  the  condition 
that  follows  the  administration  of  iron  would  seem  to  indicate  that  a 
deficient  supply  of  iron  is  the  cause  of  chlorosis,  but  numerous  ob- 
jections exist  to  this  hypothesis.  Bunge  advanced  the  idea  that  in 
chlorosis  the  iron  taken  with  the  ordinary  food  is  precipitated  in  the 
intestines  b}^  sulphides  or  other  products  of  intestinal  putrefaction, 
and  hence  there  results  a  deficiency  in  the  amount  of  iron  absorbed  and 
available  for  the  manufacture  of  hemoglobin.  ]\Iany  objections  have 
been  raised  to  tliis  hypothesis,  however,  for  competent  observers  have 
failed  to  find  any  abnormal  putrefaction  in  chlorosis,  and  others 
have  found  that  sulphide  of  iron  itself  gives  good  results  in  the  treat- 
ment of  chlorosis,  wdiile  bismuth  and  other  sulphur-binding  substances 
are  without  effect.  Furthermore,  Bunge's  contention  that  iron  ad- 
ministered in  medicinal  form  is  not  absorbed  seems  to  have  been 
completelj'-  disproved  by  several  experiments.^^ 

As  a  consequence  of  all  these  conflicting  data  we  are  at  present 
completely  in  the  dark  as  to  the  reason  for  that  failure  properly  to 
manufacture  hemoglobin  which  seems  to  be  at  the  bottom  of  chlorosis. 
The  hypothesis  that  iron  and  arsenic  favor  recovery  by  stimulating 
the  hemogenetic  tissues,  which  is  urged  by  v.  Noorden  and  others,  is 
unsatisfactory  in  the  extreme,  and  explains  nothing.  There  is  abso- 
lutely no  question  that  administration  of  iron  restores  the  composi- 
tion of  the  blood  to  normal,  usually  quite  rapidly,  and  this  seems  to 
leave  as  most  probable  the  explanation  that  in  some  way  an  iron 
starvation  is  the  fundamental  cause  of  chlorosis.  How^ever,  as  Ewing 
says,  any  theory  must  be  inadequate  that  fails  to  take  into  account 
the  age  of  puberty,  the  female  sex,  and  the  function  of  menstruation. ''° 

Pernicious  Anemia. 

In  contrast  to  chlorosis  many  evidences  of  hematolysis  may  be 
found  in  pernicious  anemia,  particularly  the  increased  amounts  of 
iron  in  the  liver,  spleen,  and  kidneys;  hemoglobinemia  and  hemoglo- 
binuria, increase  in  urobilin,  and  not  infrequently  icterus. 

Chemical  Changes."' — Erben's'-  analysis  of  the  blood  in  pernicious  anemia 
gave  the  following  results:  The  proteins  are  decreased,  both  in  the  serum'^  and 
in  the  blood  as  a  whole;  particularly  in  the  latter,  because  of  the  great  decrease  in 
the  number  of  corpuscles.  The  quantity  of  proteins  in  the  individual  corpuscles 
is  increased,  corresponding  to  their  increased  size.  Fibrin  is  decreased  in  total 
amount,  but  is  relatively  normal  as  compared  with  the  total  proteins;  albumin 

"  Full  review  with  bibliographv  bv  E.  Mever,  Ergebnisse  Physiol.,  1905  (5), 
698;  Meinertz,  Cent.  Phvsiol.  u.  Path.'  Stoffwech..  1907  (2),  652. 

^»von  Jagic  (Med.  Klin.,  1915  (11),  69)  states  that  in  chlorosis  the  Abder- 
halden  test  is  positive  with  uterine  and  ovarian  tissue. 

"  Review  and  bibliography  by  Squier,  Jour.  Lab.  Clin.  Med.,  1917  (2),  552. 

"  Zeit.  klin.  Med.,  1900  (40),  266.  Beumer  and  Biirger,  Zeit.  exp.  Path.,  1913 
(13),  343. 

"'  See  also  Heudorfer,  Zeit.  klin.  Med.,  1913  (79),  103. 

20 


306  DISTURBANCES  OF  CIRCULATION 

is  normal;  serum  globulin  much  decreased.  The  proportion  of  water  is  much 
increased,  both  in  the  serum  and  in  the  corpuscles.  Fat  is  present  in  normal 
amounts;  cholesterol  is  decreased,  although  in  relatively  normal  quantities  in 
the  corpuscles.  Lecithin  is  decreased  in  the  total  blood,  but  increased  propor- 
tionately in  the  corpuscles.  The  total  ash  is  increased,  owing  chiefly  to  an  ex- 
cessively large  proportion  of  NaCl  and  a  slight  increase  in  calcium  and  magnesium; 
potassium  and  phosphoric  acid  are  decreased  because  of  the  small  number  of  cor- 
puscles; but  the  serum  itself  contains' more  P2O5  and  potassium  than  normal. 
Although  the  total  iron  is,  of  course,  much  decreased,  there  is  iron  in  the  serum 
(indicating  hemolysis)  and  the  proportion  of  iron  in  the  corpuscles  is  increased; 
but  as  the  amount  of  iron  in  the  corpuscles  is  even  greater  than  corresponds  to 
the  hemoglobin  increase,  it  would  seem  that  either  the  hemoglobin  in  pernicious 
anemia  is  very  rich  in  iron,  or  that  the  corpuscles  contain  iron  bound  in  some  form 
other  than  hemoglobin. 

The  analyses  of  Rumpf^^  agree  quite  closely  with  those  of  Erben,  and,  taken 
jointly  with  other  analyses  in  the  literature,  show  the  large  proportion  of  water 
in  the  blood,  the  small  amount  of  solids,  the  large  amount  of  NaCl,  and  the  de- 
crease in  potassium  and  iron.  Rumpf  also  examined  the  brain,  liver,  heart,  and 
spleen  in  one  case.  Water  was  found  increased  in  the  heart,  decreased  in  the 
other  organs,  the  solids  not  being  decreased  in  any  of  the  organs.  There  was 
little  fat  in  any  of  the  organs  or  in  the  blood,  but  NaCl  was  generally  increased. 
The  liver  contained  four  or  five  times  as  much  iron  as  normal;  the  spleen  three 
or  four  times.  Rumpf  is  inclined  to  lay  great  stress  on  the  general  povertj^  of 
the  body  in  potassium,  and  suggests  its  therapeutic  application.  By  more  modern 
methods  Bloor'^  found  the  blood  lipoids  about  normal  unless  the  red  corpuscles 
were  below  50  per  cent.,  when  there  appear  high  fat  and  low  lecithin  and  choles- 
teroF'^  in  the  plasma,  but  usually  with  normal  corpuscular  lipoid  content.  The 
proportion  of  cholesterol  free  and  as  ester  is  normal.  Syllaba'''  found  bilirubin 
and  also  free  hemoglobin  in  the  blood  of  seven  patients.  Fowell'^  found  a  con- 
siderable excess  of  iron  in  the  blood  over  the  amount  combined  with  hemoglobin. 
Schumm^^  could  find  no  proteoses  or  other  evidences  of  protein  decomposition  in 
the  blood  in  a  case  of  pernicious  anemia,  but  he  did  find  free  hematin.*"  The 
tendency  to  hemorrhage  observed  in  this  disease  may  depend  on  a  slight  decrease 
in  the  prothrombin  and  a  reduction  in  the  number  of  platelets.  ^1 

V.  Jaksch  and  also  v.  Limbeck^^  have  found  some  decrease  in  total  alkalinity, 
which  probably  depends  on  the  loss  of  proteins  and  their  fixed  alkali.*^  The  red 
corpuscles  are  very  susceptible  to  hemolysis  by  lowering  of  osmotic  pressure 
("high  isotonicity,"  equal  to  0.54  per  cent.  NaCl — v.  Limbeck).  The  specific 
gravity  of  the  whole  blood  is,  of  course,  decreased,  but  the  corpuscles  themselves 
have  practically  normal  specific  gravity,  while  the  decrease  is  chiefly  in  the  serum. *^ 
Bile  pigment  is  frequently  found  in  the  blood,  but  so  bound  that  it  does  not  escape 
into  the  urine  and  it  does  not  always  cause  evident  jaundice.  Bile  salts  may  be 
found  in  the  blood,  either  with  or  without  pigment  (Blankenhorn).*^  There  is  a 
marked  increase  in  the  urobilin  output,  corresponding  in  degree  to  the  amount 
of  hemolysis. ^'^ 

In  six  cases  of  pernicious  anemia  Stiihlen^^  found  abundant  iron  in  the  liver 
and  spleen  microscopicallj^,  and  less  constantly  in  the  kidneys  and  bone-marrow. 

^*  Berl.  klin.  Woch.,  1901  (38),  477;  !^ee  also  Kahn  and  Barsky,  Arch.  Int.  Med., 
1919  (23),  334, 

"  Jour.  Biol.  Chem.,  1917  (31),  79. 

^«  Corroborated  by  Pacini,  Amcr.  Med.,  1918  (13),  92. 

"  Abst.  in  Folia  Hematol.,  1904  (1),  283  and  589. 

^8  Quart.  Jour.  Med.,  1913  (6),  179. 

"  Hofmeister's  Beitr.,  1903  (4),  453. 

soZeit.  physiol.  Chem.,  1910  (97),  32. 

81  Drinker  and  Hurwitz,  Arch.  Int.  Med.,  1915  (15),  733. 

»'^"Klin.  Pathol,  des  Blutes,"  Jena,  189(1,  p.  311. 

83  See  Brandenburg,  Zeit.  klin.  Med.,  1902  (45),  157. 

8"  Bonninger,  Zeit.  exp.  Path.,  1912  (11),  1. 

85  Arch.  Int.  Med.,  1917  (19),  344. 

8"  See  Ifansnuuui  and  Howard,  Jour.  Anier.  Med.  Assoc,  1919  (73),  1262. 

8^  Deut.  Arch.  klin.  Med.,  1895  (54),  248  (literature). 


PERNICIOUS  ANEMIA  307 

Hunter**  gives  the  following  results  of  analysis  of  the  liver,  kidney,  and  spleen 
for  iron: 

Liver  and 

kidney.  Spleen. 

Pernicious  anemia,  seven  cases  average 0.300  per  cent.  0. 12;'>  per  cent. 

Other  conditions  (with  anemia),  average 0.079  per  cent.  0.362  per  cent. 

Health}'  organs 0.084  per  cent.  0.090  per  cent. 

Iron  is  also  found  in  the  hemolymph  glands,  sometimes  more  abundantly  than  in 
the  spleen  (Warthin).*^ 

Extensive  studies  on  the  protein  metabolism  of  pernicious  anemia  by  Rosen- 
quist'"  showed  that  there  is  a  considerable  destruction  of  tissue  proteins,  as  indi- 
cated by  nitrogen  loss,  but  that  at  times  nitrogen  may  be  stored  up  for  brief 
periods.  At  times  there  may  also  be  an  excessive  elimination  of  purine  nitrogen, 
indicating  destruction  of  nuclear  elements.  Calorimetric  studies  show  the  metab- 
olism to  be  slightly  above  normal.^'  In  anemia  due  to  Bothriocephalus  quite 
similar  changes  were  observed. 

Hunter^-  describes  the  condition  of  the  urine  in  pernicious  anemia,  particularly 
with  reference  to  the  elimination  of  much  ''pathological  urobilin,"^'  which  seems 
to  be  produced  by  intracellular  destruction  of  hemoglobin.  Iron  may  also  appear 
in  the  urine  in  increased  quantities."  There  is  an  increased  elimination  of  oxy- 
proteic  acid  nitrogen,  other  urinar}'  nitrogen  constituents  being  normal  (Kahn 
and  Barsky)."^  No  constant  metabolic  changes  follow  splenectomy  in  pernicious 
anemia.'^ 

Summary.^*^ — Putting  together  the  above  findings,  we  see  tliat  in 
pernicious  anemia  we  have  everj^  evidence  that  excessive  hemolysis 
is  taking  place,  and  the  fact  that  continued  poisoning  by  toluylendia- 
mine^^  and  other  hemolytic  poisons,  such  as  that  of  Bothriocephalus ,^^ 
may  give  rise  to  a  condition  resembling  pernicious  anemia  very  closely, 
indicates  strongly  that  hemolytic  poisons  are  the  cause  of  pernicious 
anemia.  Histological  studies  show  the  same  thing,  and,  as  Warthin^^ 
says:  "The  hemolj^sis  of  pernicious  anemia  does  not  differ  in  kind 
from  that  occurring  normally  or  in  certain  diseased  conditions;  the 
difference  is  one  of  degree  only."  The  hemolysis  seems  to  go  on  chiefly 
inside  of  phagocytic  cells  instead  of  in  the  blood,  probably  because  the 
phagoc3'tes  pick  up  the  corpuscles  as  soon  as  they  have  been  injured 

8«  Lancet,  1903  (i),  283;  similar  results  obtained  bv  Evffel,  Jour.  Path,  and 
Bact.,  1910  (14),  411. 

89  .\mer.  Jour.  Med.  Sci.,  1902  (124),  674. 

90  Zeit.  klin.  Med.,  1903  (49),  193  (literature).  See  also  Minot,  Bull.  Johns 
Hopkias  Hosp.,  1914  (25),  338. 

91  Mever  and  DuBois,  Arch.  Int.  Med.,  1916  (17),  ^65;  Grafe,  Peut.  Arch, 
klin.  Med.,  1915  (118),  148.  See  also  Tompkias  et  al.,  Arch.  Int.  Med..  1919 
(23),  441. 

92  British  Med.  Jour.,  1890  (ii),  1  and  81. 

93  See  also  Mott,  Lancet,  1890  (1),  287;  and  Syllaba,  Abst.  in  FoUa  Hematol., 
1904  (1),  283. 

9^  Keunerknecht,  Virchow's  Arch.,  1911  (205),  89.  Not  confirmed  by  Quecken- 
stedt.  Zeit.  klin.  Med.,  1913  (79\  49;  bibliography. 

95  Denis,  Arch.  Int.  Med.,  1917  (20),  79. 

9^  Review  on  etiology  of  pernicious  anemia  given  bv  Vogel,  Jour.  Amer.  Med. 
Assoc,  1916  (66),  1012. 

9"  Syllaba,"  Hunter**  (loc.  cit.). 

9*  In  horses  a  condition  resembling  pernicious  anemia  seems  to  be  produced 
by  a  toxic  product  of  the  larva;  of  a  fly.  Oestrus  cqui,  which  is  found  in  the  walls 
of  the  stomach  of  anemic  horses  (Seyderhelm,  Arch.  exp.  Path.  u.  Pharm.,  1914 
(76),  149). 


308  DISTURBANCES  OF  CIRCULATION 

by  the  hemolytic  poisons.  In  some  instances  cholesterol  administra- 
tion improves  the  anemia,  which  suggests  that  the  poison  attacks 
the  lipoids  of  the  corpuscles,^'  as  so  many  hemolytic  agents  do.  Both- 
riocephalus  anemia,  which  so  closely  resembles  the  "pernicious"  form, 
seems  to  be  caused  by  a  hemolytic  lipoid,  ^  presumably  a  cholesterol 
ester  of  oleic  acid,  and  there  is  a  growing  tendency  to  associate  hemoly- 
tic lipins  with  the  etiology  of  pernicious  anemia.^  However,  although 
in  hemolytic  anemias  there  is  an  increased  amount  of  unsaturated 
lipins  in  the  blood^  Medak*  did  not  find  the  isolated  lipoids  to  be 
particularly  hemolytic.^  (See  Hemolysis,  Chapter  ix.)  The  origin 
and  the  nature  of  the  specific  hypothetical  poisons  have  been  sought 
in  vain.  Some  authors  have  referred  them  to  infections  of  unknown 
nature,  occurring  perhaps  in  the  mouth  and  gastrointestinal  tract 
( Hunter), ^^  or  to  hemolytic  products  of  intestinal  putrefaction,^  or  to 
faulty  metabohsm.  For  example,  Iwao^  has  found  that  tyramine 
(p-oxyphenyl-ethylamine)  produces  in  guinea  pigs  a  blood  picture 
resembling  pernicious  anemia,  and  this  amine  ma 5^  be  produced  either 
in  the  intestines  or  during  metabolism.  Others,  with  perhaps  the  best 
of  grounds,  would  ascribe  pernicious  anemia  to  a  multiplicity  of  causes, 
which  produce  a  protracted  slight  hemolysis  that  continues  until  the 
hematogenetic  organs  give  out,  their  exhaustion  being  perhaps  hastened 
by  the  influence  of  the  toxic  substances  in  the  blood;  hematogenesis 
then  becomes  insufficient  to  replace  the  lost  corpuscles,  and  the  picture 
of  pernicious  anemia  is  established.^  The  relatively  great  proportion 
of  the  iron  that  is  stored  in  the  liver  supports  the  view  that  the 
hemolysis  takes  place  in  portal  territory,  and  many  other  facts  point 
to  the  same  conclusion,  but  it  is  not  generally  accepted  that  the 
spleen  plays  an  essential  role  in  causing  pernicious  anemia  through 
excessive  phagocytosis  or  production  of  hemolytic  poisons.' 

»^  See  Reicher,  Bed.  klin.  Woch.,  1908  (45),  1838. 

1  Tallquist,  Zeit.  klin.  Med.,  1907  (61),  427;  Arch.  exp.  Path.  u.  Pharm.,  1907 
(57),  367. 

^  SeeLiidke  and  Fejes,  Deut.  Arch.  klin.  Med.,  1913  (109),  433. 

3  See  King,  Arch.  Int.  Med.,  1914  (14),  145;  Csonka,  Jour.  Biol.  Cheni.,  191S 
(33),  401. 

■»  Biochem.  Zeit.,  1914  (59),  419. 

«  See  McPhedran,  Jour.  Exp.  Med.,  1914  (18),  527. 

^  See  Kiilbs  (Arch.  exp.  Path.  u.  Pharm.,  1906  (55),  73),  who  found  the  in- 
testinal contents  of  patients  with  chronic  intestinal  disorders  to  contain  hemo- 
lytic substances  of  undetermined  character.  IfcMiiolytic  lijioids  in  the  intestinal 
contents  have  been  described  by  Bergor  and  Tsuchiga  (Deut.  Arcli.  klin.  Med., 
1909  (96),  252)  and  Liidke  and  Fejes,  loc.  cit.;  but  this  observation  failed  of  con- 
firmation by  Ewald  (Deut.  med.  Woch.,  1913  (39),  1293). 

Herter  (Jour.  Biol.  Chem.,  190i)  (2),  1)  suggested  a  relation  between  intestinal 
infection  with  B.  acrocjencR  capsulatiits,  wliich  produces  hemolytic  substances,  and 
pernicious  anemia. 

^  Biochem.  Zeit.,  1914  (59),  436. 

'^  Sec  also  limiting,  .Johns  Hopkins  llosp.  Bull.,  1905  (16),  222;  Pai)penheim, 
Folia  Serologica,  1910  (10),  217. 

«See  Hirschlield,  Zeit.  klin.  Med.,  1919  (87),  165. 


DISEASES  OF  THE  BLOOD  30'.) 

Leukemia 

In  Icukoinia  the  chciiiical  changes  in  the  red  corpuscles  take  a  less 
prominent  position,  resenil)ling  cither  those  of  a  secondary  anemia 
or  chlorosis,  while  the  enormous  numl)er  of  leucocytes  is  the  prominent 
feature  and  causes  marked  alterations  in  the  composition  of  the  l)lood. 
Large  quantities  of  nucleoprotcins  and  also  of  the  intracellular  enzymes 
are  introduced  into  the  blood  by  the  excessive  leucocytes.  As  the 
leucocytes  are  constantly  breaking  down,  more  or  less  of  the  products 
of  their  decomposition  are  present  in  the  blood  and  appear  in  the  urine. 
Because  of  the  relatively  slight  metabolic  activity  of  the  lymphocytes 
tiio  various  chemical  alterations  are  all  less  marked  in  Ijmiphatic  than 
in  myelogenous  leukemia.^"  There  is  a  notable  reduction  in  antibody 
production  in  leukemia, ^^  presumably  because  of  the  changes  in 
the  bone  marrow;  it  is  said  that  typhoid  infection  in  leukemics  may  fail 
to  result  in  agglutinin  formation. 

Chemistry  of  the  Blood.— Co n.sidering  the  quantitative  alterations  in  the  con- 
stituents of  the  blood,  we  find  the  specific  gravity  lowered,  but  not  so  much  as 
itjwould  be  in  a  simple  anemia  with  equally  low  hemoglobin,  for  the  loss  of  hemo- 
globin is  partly  compensated  bj"  the  increase  in  leucocytes  and  their  products. 
Fibrinogen  is  usually  increased  in  myelogenous  leukemia.'-  The  serum  shows 
but  slight  change  in  specific  gravity,  a  slight  decrease  in  proteins'^  being  com- 
pensated by  an  increase  in  the  NaCl.  The  freezing-point  of  the  blood  is  lowered 
(Cohn^*),  which  is  probably  due  to  the  increase  in  crystalloidal  products  of  cel- 
lular decomposition.  Erben"  found  that  in  lymphatic  leukemia  the  serum 
contains  less  cholesterol  than  normal,  although  the  fat  content  may  be  rather 
high.  Calcium  is  frequently  found  increased,  probably  because  of  destruction 
of  the  bone  tissue.  In  the  red  corpuscles  the  proportion  of  iron,  protein  and 
potassium  is  decreased  as  is  also  that  of  the  cholesterol,  that  of  the  lecithin  and 
water  being  somewhat  increased.  The  total  amount  of  potassium  and  iron  in  the 
blood  is  decreased,  but  the  P2O5  in  the  ash  is  increased  because  of  the  large  amount 
of  nucleoprotein  in  the  blood.  A  number  of  the  earlier  writers  describe  a  decreased 
alkalescence  which  probably  is  due  to  the  deficiency  in  the  fLxed  alkali  of  the  pro- 
teins.    There  is  an  increased  excretion  of  iron  in  the  urine  and  feces.'"' 

The  poor  coagulation  0/  leukemic  blood  has  long  been  known,  but  the  reason 
for  it  has  not  yet  been  ascertained.  Some  investigators  have  reported  a  deficiency 
in  fibrin,  while  others  have  found  it  increased.  More  recent  reports,  however, 
indicate  that  there  is  no  marked  change  in  either  the  amount  of  fibrinogen  or  of  the 
fibrin-ferments.  Erben'-  found  a  normal  amount  of  fibrin  in  the  blood  in  lym- 
phatic leukemia;  and  in  three  cases  of  myelogenous  and  one  of  lymphatic  leukemia, 
Pfeiffer'"  found  the  amount  of  fibrinogen  nearly  normal.  This  is  quite  remarkable 
in  view  of  the  fact  that  in  ordinary  forms  of  leucocytosis  both  the  amount  of  fibrin- 
ogen and  the  rapidity  of  clotting  are  increased.     It  is,  therefore,  extremely  difficult 

1°  Stern  and  Eppenstein  have  observed  that  the  striking  proteolytic  power  of 
the  leucocytes  from  the  blood  in  myelogenous  leukemia  is  not  shown  by  the 
leucocytes  in  lymphatic  leukemia  (Sitz.  d.  Schles.  Ges.  f.  vaterland.  Kultur, 
June  29,  1906). 

"  Rotky,  Zent.  inn.  Med.,  1914  (35),  953. 

12  Erben,  Zeit.  klin.  Med.,  1908  (66),  278;  full  details  on  composition  of  the 
blood  in  leukemia. 

"  Little  change  was  found  in  the  protein  content  of  the  serum  by  Heudorfer.. 
Zeit.  klin.  Med.,  1913  (79),  103. 

"Mitteil.  aus  dem  Grenzgeb.  Med.  u.  Chir.,  1906  (15),  H.  1. 

1*  Zeit.  klin.  Med.,  1900  (40),  282. 

'«Kennerknecht,  \  irchow's  Arch.,  1911  (205),  89. 

"  Cent.  f.  inn.  Med.,  1904  (25),  809. 


310  DISTURBANCES  OF  CIRCULATION 

to  understand  the  poor  coagulability  of  leukemic  blood,  but  study  of  the  factors  of 
coagulation  by  modern  methods  may  clear  this  up,  for  in  one  case  so[studied  VSTiip- 
ple'^  found  an  increase  in  antithrombin. 

Decomposition  Products. — Of  particular  interest  is  the  finding"  in  the  blood 
of  decomposition  products  of  the  leucocytes,  which  are  probably  produced  by 
autolysis  of  the  leucocytes.  (See  Leucocytic  Enzymes,  Chapter  iii.)  Normal 
leucocytes  are  rich  in  autolytic  enzymes,  which  under  ordinary  circumstances  seem^ 
to' be  held  in  check  by  the  antienzymes  of  the  blood.  In  leukemia  this  antienzyme 
action  seems  to  be  insufficient  to  prevent  leucocytic  autolysis,  for  even  in  freshly 
drawn  blood  proteoses  for  at  least  non-coagulable  proteins)  may  be  present.'' 
According  to  Erben,  this  is  true  only  of  myelogenous  leukemia,  the  fresh  blood  in 
lymphatic  leukemia  not  only  being  free  from  non-coagulable  protein,  but  further- 
more this  product  of  proteolysis  does  not  soon  develop  when  the  blood  is  kept 
aseptically  at  incubator  temperature.  This  is,  of  course,  what  one  wouldi  expect 
in^view  of  the  well-known  enzyme-richness  of  the  polymorphonuclear  leucocj'tes 
neutrophile  cells  seem  to  be  the  chief  source  of  proteoses,  since  their  granules  soon 
and  the  scarcity  of  proteolytic  enzymes  in  lymphocytes.  Erben  states  that  the 
disappear  in  blood  that  is  undergoing  autolysis,  whereas  the  eosinophiles  preserve 
their  granules  well,  and  true  proteoses  are  not  present  in  blood  rich  in  mast  cells 
(i.  e.,  myeloma).  The  marrow,  spleen  and  lymph  glands  are  found  strongly  pro- 
teolytic (according  to  the  plate  method),  in  myelogenous  leukemia,  but  in  lym- 
phatic leukemia  and  pseudoleukemia,  only  the  marrow  shows  a  slight  acti^Hty.-<' 
Schumm^i  found  in  the  blood  in  a  case  of  myelogenous  leukemia  several  varieties 
of  proteoses,  most  abundant  being  the  so-called  deutero-albumose;  in  another  he 
also  found  peptone,  leucine,  and  tyrosine.  In  addition  he  demonstrated  the  auto- 
lytic nature  of  the  changes  that  occur  in  leukemic  blood  after  death  (see  also 
"Autolysis  in  Leukemia,"  Chap.  iii).  Most  observers  have  failed  to  find  alhumose 
in  the  urine  in  leukemia.  Because  of  the  involvement  of  the  bone  marrow,  small 
amounts  of  Bence-Jones  protein,  as  well  as  Morner's  bodj"-,  may  be  found  in  the 
urine. -^  Kolisch  and  Burian^^  not  only  found  nucleoprotein  constantly,  and  albu- 
mose  frequently,  but  in  one  case  of  lymphatic  leukemia  they  found  histon  in  the 
urine,  which  undoubtedly  came  from  nucleoprotein  decomposition. 

The  oxidase  reaction  is  conspicuous  in  certain  of  the  cells  of  myeloid  leukemia, 
especially  the  large,  non-granular  cells  of  acute  leukemia,-*  but  it  is  not  known 
that  these  oxidases  influence  the  chemistry  of  the  disease.  In  spite  of  the  richness 
of  leucocytes  in  lipases  the  serum  shows  no  increased  lipolytic  activity.-^ 

Protein  Metabolism. — Stejskal  and  Erben-*^  studied  the  metabolism  of  a  case 
of  myelogenous  and  of  a  case  of  lymphatic  leukemia,  and  found  the  nitrogen  loss 
much  greater  in  the  myelogenous  form,  although  food-absorption  was  better  than 
in  the  lymphatic ;  they  consider  that  protein-destroying  forces  are  at  work  in  myelo- 
genous leukemia,  similar  to  those  of  cancer  cachexia,  so  that  nitrogenous  equi- 
librium cannot  be  attained. 

As  the  most  characteristic  products  of  decomposition  of  nucleoproteins  are 
the  purine  bases,  one  would  also  expect  to  find  them  present  in  leukemia,  and  early 
writers  mention  the  finding  of  purine  bases  and  uric  acid  in  the  blood  and  spleen. 
The  urinary  findings  in  this  respect  have  been  very  variable.  Ebstein"  observed 
the  complication  of  leukemia  with  gout  which  he  considered  a  coincidence,  and 
also  noted  uric-acid  concretions  in  the  urinary  passages  in  four  cases.  Numerous 
other  authors  have  described  increased  uric-acid  elimination,  while  some  have 
observed  increase  in  the  purine  bases,  either  with  or  without  uric-acid  increase. 
Magnus-Levy^^  observed  a  particularly  large  uric-acid  output  in  acute  leukemias, 

i«  Arch.  Int.  Med.,  1913  (12),  637. 

19  For  literature  see  Erben,  Zeit.  f.  Ileilk.  (Int.  Mod.  Abt.),  1903  (24),  70. 

20  Jochmann  and  Zicglor,  Mlinch.  med.  Woch.,  190G  (53),  2093. 

2'  Ilofmeister's  Hcitr.,  1903  (4),  442;  Dcut.  med.  Woch.,  1905  (31),  183. 

"  Boggsand  (iuthrie,  Hull.  .lohns  IIoi)kins  Hosp.,  1913  (24),  3GS. 

23  Zeit.  kliii.  Med.,  l.Si)G  (29),  374  (literature  on  albuminuria  in  leukemia). 

"  Dunn,  Ouart.  Jour.  Med.,  1913  (ti),  293. 

"  Caro,  Zeit.  klin.  Med.,  1913  (78),  28(j. 

"Zeit.  f.  klin.  Med.,  1900  (39),  151. 

"  For  lit(M-ature  see  resume  1)V  Walz  in  Cent.  f.  Patiiol.,  1901  (12),  985. 

2sVirchow's  Arch.,  189S  (152),  107. 


LEUKEMIA  'A  1 1 

but  also  fouiul  that  the  relation  be.  ween  the  number  of  leii(;ocytc.s  and 
the  utif  acid  is  extreincly  variaI)Io.  Soiuctiincs  the  iiitroKen  loss  is  very 
great — even  as  much  as  20  ^m.  per  day — and,  corresponding  with  the  destruction 
of  nucleoj)roteins  and  the  resulting  uric-acid  formation,  phosphoric-acid  excretion 
is  often  greatly  increased — even  up  to  15  gm.  ])er  day.  On  the  other  hand,  the 
results  obtained  by  many  other  writers  have  been  in  every  respect  extremely  varia- 
ble; some  have  found  no  increase  in  uric  acid,  some  even  report  a  decrease;  likewise 
the  P2O;,  has  been  found  even  less  than  normal.  For  example,  in  a  carefully  studied 
case  of  lymphatic  leukemia,  Henderson  and  Edwards'-"-'  found  during  six  months 
no  excessive  excretion  of  uric  acid  or  phosphoric  acid.  Zalesky  and  Erben  found 
likewise  no  coasidcrable  increase  in  the  uric  acid  in  lymphatic  leukemia,  but  in 
myelogenous  leukemia  the  uric  acid  was  much  increased;  on  the  other  hand,  the 
amount  of  elimination  of  purine  bases  was  reversed  in  the  two  forms,  and  creatin 
was  decreased  in  both.  Lipstein'°  found  no  excessive  elimination  of  amino-acids 
even  in  myelogenous  leukemia.  An  increase  in  calcium  is  quite  constantly  ob- 
served, and  attributed  to  the  bone  destruction-^  occurring  in  this  disease. 

Undoubtedly  these  variations  in  results  depend  upon  the  known  fluctuations  in 
the  course  of  the  pathological  processes  of  leukemia;  the  number  of  leucocytes,  the 
size  of  the  lymphatic  organs,  and  the  general  condition  of  the  patient  all  vary 
greatly  from  time  to  time,  often  with  remarkable  rapidity,  and  the  excretion  of 
products  of  metabolic  activity  must  vary  likewise.  It  can  hardly  be  questioned 
that  the  enormous  increase  in  the  amount  of  lymphoid  tissue  in  the  body  and 
blood  must  give  rise  to  a  greatly  increased  nuclein  catabolism,  with  consequent 
appearance  of  its  products  (uric  acid,  purine  bases,  and  phosphoric  acid)  in  the 
urine.  This  seems  to  be  well  demonstrated  by  the  increased  elimination  of  uric 
acid  and  purine  bases,  together  with  a  general  increase  in  the  nitrogen  output  that 
has  been  frequently  observed  following  the  therapeutic  use  of  a:-rays  in  leukemia, 
which  is  attributed  to  the  increased  autolysis  that  x-rays  are  known  to  produce. 
Radium  has  a  similar  effect,  increasing  enormously  the  urinary  total  nitrogen,  urea, 
ammonia,  less  markedly  the  uric  acid,  but  especially  the  phosphates.^' 

According  to  Rosenstern^'-  the  x-rays  affect  chiefly  the  leucogenic  tissues  rather 
than  the  adult  leucocytes.  Lipstein  also  found  an  excessive  elimination  of 
amino-acids,  in  the  urine  of  leukemic  patients  treated  by  .c-rays.^^  According  to 
Curschmann  and  Gaupp,^*  the  blood  of  leukemic  patients  who  have  been  exposed 
to  x-rays  contains  a  specific  leucocytotoxin,  which  may  be  produced  by  a  process  of 
autoimmunization  against  the  leucocytic  substance  set  free  by  the  disintegrated 
leucocytes.  Capps  and  Smith^^  have  obtained  similar  results.  A'-rays  seem  not 
to  alter  the  total  metabolism  appreciably.''^ 

Charcot's  crystals  (also  called  Charcot-Ley den  and  Charcot-Neumann  crystals) 
represent  a  peculiar  and  striking  product  of  nuclear  destruction  that  has  fre- 
quently^ been  found  associated  with  leukemia.  These  crystals  were  first  observed 
by  Robin3  7(1853)  in  leukemic  tissues,  but  have  been  named  after  Charcot,  who, 
with  Robin,  described  their  properties.  They  were  described  by  Charcot  as  color- 
less, refractile,  elongated  octahedra;  insoluble  in  alcohol,  ether,  and  glycerol; 
soluble  in  hot  water,  acids,  and  alkalies;  size  variable,  from  0.016  by  0.005  mm.  up. 
These  crystals  have  been  found  not  only  in  the  tissues  and  blood  of  cadavers,  but 

23  Amer.  Jour,  of  Physiol.,  190.3  (9),  417. 

30  Hofmeister's  Beitr.,  1905  (7),  527. 

31  Knudsonand  Erdos,  Boston  Med.  Surg.  Jour.,  1917  (176),  503. 

32  Miinch.  med.  Woch.,  1906  (53),  1063. 

"  Literature  on  effects  of  x-rays  in  leukemia,  see  Arneth,  Berl.  klin.  Woch., 

1905  (42),  1204;  Musser  and  Edsall,  Univ.  of  Penn.  Med.  Bull.,  1905  (18),  174; 
Rosenberger,   Miinch.  med.   Woch.,    1906   (53),   209;  Williams,   Biochem.   Jour., 

1906  (1),  249;  Lessen  and  Moraw^tz,  Deut.  Arch.  klin.  Med.,  1905  (83),  288; 
Koniger,  Deut.  Arch.  klin.  Med.,  1906  (87),  31. 

34  Miinch.  med.  Woch.,  1905  (52),  2409. 

36  Jour.  Exp.  Med.,  1907  (9),  51;  see  also  Klieneberger  u.  Zoeppritz,  Miinch. 
med.  Woch.,  1906  (53),  No.  18;  Milchner  u.  Wolff,  Berl.  klin.  Woch.,  1906  (43), 
No.  23. 

36  Arch.  Int.  Med.,  1917  (19),  890. 

3'^  Literature  given  by  v.  Leyden,  Festschrift  fiir  Salkowski,  Berlin,  1904,  p.  1. 


312  DISTURBANCES  OF  CIRCULATIOX 

also  occasionally  in  the  freshly  drawn  blood  of  leukemics.  Poehl^s  believes  thorn 
to  be  the  same  as  Bottcher's  spermin  crystals,  and  derived  from  decomposed 
nucleins.  Schreiner  considers  that  these  spermin  crystals  are  phosphoric  acid 
salts  of  spermin  (C2H5N),  or,  as  Majert  and  Schmidt  give  it,  C^HioXo,  with  the 

structure  UN  <nH"_r'TT^>  NH,  thus  being  similar  to,  although  not  identiral 

with,  piperazin.  The  entire  question  of  the  composition  of  spermin  is  still  un- 
settled, ^^  however;  and  it  is  probable,' furthermore,  that  the  crystals  found  in 
leukemia  are  not  identical  with  the  crystals  observed  in  semen. 

Crystals  that  appear  similar  are  also  found  in  asthmatic  sputum,  empyema, 
and  ascites  fluid,  bone-marrow,  and  tumors,  and  it  has  been  suggested  that  they 
are  derived  from  or  related'  to  the  oxyphile  granules  of  the  eosinophiles.*"^  This 
view  implies  an  agreement  with  Gumprecht's  opinion  that  the  crystals  seen  in 
bone-marrow,  asthmatic  sputum,  etc.,  are  not  spermin,  but  of  protein  nature. 
As  can  be  seen,  the  nature  and  significance  of  Charcot's  crystals  are,  at  the  pres- 
ent time,  quite  undetermined. 

Summary. — The  chemical  changes  observed  in  leukemia  depend 
upon  the  excessive  quantity  of  leucocytes  and  lymphoid  tissue,  which 
undergo  processes  of  disintegration  at  irregular  intervals,  with  the 
result  that  the  products  of  nucleoprotein  destruction  (uric  acid,  purine 
bases,  and  phosphoric  acid)  appear  in  the  urine  in  increased  quantities. 
As  the  large  neutrophiles  contain  abundant  autolytic  enzymes,  the  prod- 
ucts of  cell  autolysis  (proteoses,  amino-acids,  and  products  of  nucleo- 
protein destruction)  may  appear  at  times  in  the  urine  and  in  the  blood; 
because  of  the  small  amount  of  such  enzymes  in  the  lymphocytes,  these 
changes  are  all  much  less  marked  in  lymphatic  leukemia.  Charcot's 
crystals,  which  are  perhaps  derived  from  leucocytic  nucleoproteins, 
may  be  found  in  the  blood  and  tissues.  The  changes  in  the  red  cells 
are  chiefly  those  of  a  secondary  anemia,  with  occasionally  some  chlo- 
rotic  features.  The  chemical  findings  of  leukemia  throw  no  light 
whatever  upon  the  cause  of  the  disease. 

Pseudoleukemia  and  Hodgkin's  disease  show  only  the  evidences  of 
a  secondary  anemia,  without  the  chemical  changes  of  either  leukemia 
or  pernicious  anemia.  There  seems  to  have  been  little  study  of  tlu' 
chemical  processes  of  these  diseases.  Moraczewsld^i  reports  a  study 
of  metabohsm  in  one  case,  designated  by  him  as  pseudoleukemia  and 
so  quoted  in  subsequent  literature,  although  the  only  leucocyte  count 
mentioned  in  the  original  article  was  171,000.  This  case  showed  some 
retention  of  nitrogen  and  calcium,  with  little  change  in  the  phosphorus 
and  purine  bases  in  the  urine. 

HYPEREMIA 
Active  Hyperemia 

This  condition  is  associated  with  but  few  chemical  changes.  Cei- 
tain  chemicals  may  cause  active  hyjxM-emia;  some  locally,  as  in  the 

"  Deut.  med.  Woch.,  1895  (21),  475. 

^*  Literature,  see  HaTiimarsten,  Amer.  Transl.,  lUOl,  p.  -120. 
■•»  Literature,  see  Floderer,  Wien.  klin.  Woch.,  190:i  (16),  276;  Predtetschenskv, 
Zeit.  klin.  Med.,  1<)0()  (5«)),  29. 

*'  Virchow's  .Vrch.,  1898  (151),  22. 


HYPEREMIA  313 

case  of  irritants,  such  as  alcohol,  ether,  ammonia,  mustard,  etc.,  which 
act  neither  by  producing;  a  local  vasodilator  stimulus  or  Ijy  paralyzing 
the  vasoconstrictors.  Other  substances  may  produce  active  hypere- 
mia in  special  vascular  areas,  e.  gr.,  cantharides  causes  active  hyperemia 
in  the  kidneys,  probably  because  of  its  elimination  through  these 
organs;  pilocarpin  causes  active  hyperemia  in  the  salivary  glands  and 
skin,  which  is  associated  with  increased  function.  In  general,  func- 
tional activity  is  associated  with  active  hyperemia,  and  GaskelP^  has 
suggested  that  this  is  due  to  atonicity  of  the  vascular  muscle,  the  result 
of  decreased  alkalinity  of  the  lymph  flowing  away  from  the  active  organ 
along  the  vessel-walls,  it  having  been  found  that  alkalies  cause  a  tonic 
contraction  and  acids  an  atonic  dilation  of  arterial  muscle.  '" 

Pathological  active  hyperemia  is  seldom  of  long  enough  duration 
to  lead  to  any  alterations  in  the  tissues  in  which  it  occurs.  The  blood 
itself  remains  unchanged,  except  that  the  venous  blood  going  from  the 
part  contains  much  less  CO2  and  more  oxygen  than  usual,  because 
more  oxygen  is  brought  to  the  tissues  than  can  be  used.'*^ 

Passive  Hyperemia 

Passive  hyperemia  is  almost  equally  unassociated  with  chemical 
changes,  especially  in  its  etiology,  as  it  depends  solely  upon  mechan- 
ical factors.  Some  chemical  alterations  result,  however,  from  the 
changes  in  the  stagnating  blood,  wliich  may,  if  the  obstruction  to  out- 
flow is  severe,  become  of  venous  character  in  the  capillaries  of  the  con- 
gested area.  Oxidation  in  the  tissues  is,  therefore,  impaired,  and  some 
fatty  changes  may  result,  e.  g.,  in  the  center  of  congested  liver  lobules. 
Waste  products  accumulate,  and  possibly  noxious  products  of  meta- 
bolism are  formed  under  lack  of  oxidation;  either  from  these  causes  or 
solely  from  pressure  and  lack  of  nutrition  there  is  a  tendency  to  atrophy 
of  the  more  specialized  parenchymatous  cells,  and  a  proliferation  of 
connective  tissues.  The  atrophy  of  parenchyma  is  seen  particularly 
in  the  liver,  the  increase  of  connective  tissue  in  the  spleen. ^^  In  the 
Ividney  neither  atrophy  nor  stroma  proliferations  are  pronounced,  but 
the  renal  function  is  greatly  impaired,  since  it  depends  upon  the 
amount  and  quality  of  the  blood  brought  to  the  kidney.""^  Whether 
connective-tissue  proliferation  in  hyperemia  depends  upon  overnutri- 
tion  or  upon  irritation  by  waste-products,  or  is  compensatory  to  par- 
enchymatous atrojihy,  may  be  looked  upon  as  still  an  open  question. 

*^  Quoted  by  Lazarus-Barlow,  "Manual  of  General  Pathology,"  1904,  p. 126. 

"See  discussion  by  WooUey,  Jour.  Amer.  Med.  Assoc,  1914  (63),  2279;  and 
by  Adler,  Jour.  Pharm.,  1916  (8),  297. 

"  Polycythemia  (Vaquez-Osler  disease)  is  accompanied  by  an  increase  in  the 
total  nitrogen  of  the  blood,  in  proportion  to  the  number  of  erythrocytes;  but  the 
nitrogen  content  of  the  individual  erythrocyte  is  decreased,  (v.  Jaksch.  Zent. 
inn.  Med.,  1912  (33),  397). 

*^  See  Christian,  Jour.  Amer.   Med.  Assoc,   1905  (45),   1615. 

"  See  Rowntree  and  Geraghty,  Arch.  Int.  Med.,  1913  (11),  121;  Nonnenbruch, 
Deut.  Arch.  klin.  Med.,  1913  (110),  162. 


314  DISTURBANCES  OF  CIRCULATION 

Probably  only  the  first  two  factors  apply  to  the  connective-tissue 
growth  observed  in  the  congested  spleen,  the  clubbing  of  the  fingers 
in  congenital  heart  disease,  or  the  thickening  of  the  subcutaneous 
tissues  in  passive  congestion  of  the  lower  extremities. 

Changes  in  the  Blood. — Venous  blood  differs  from  arterial,  not 
only  in  its  increased  load  of  CO2  and  other  waste  products,  but  also 
in  other  ways.  Venous  blood  generally  clots  less  readily  than  arterial 
blood. *^  It  contains  more  diffusible  alkali  because  the  CO2  combines 
with  and  tears  away  part  of  the  bases  that  are  held  by  the  proteins, 
especially  in  the  corpuscles,  and  so  alkaline  carbonates  are  formed 
and  enter  the  plasma.  Blood  from  the  jugular  vein  on  this  account 
contains  20-25  per  cent,  more  diffusible  alkali  than  carotid  blood 
(Hamburger).'*^  Since  the  bactericidal  power  of  the  blood  has  been 
shown  to  increase  directly  with  the  alkalinity,  this  property  may  be 
of  importance  in  pathology.  For  example,  the  relative  infrequency 
of  infections  in  the  right  side  of  the  heart  may  not  depend  solely  upon 
lessened  liability  to  endocardial  damage,  as  generally  considered,  but 
is  possibly  due  in  part  to  the  greater  bactericidal  power  of  venous 
blood.  The  same  property  probably  explains  the  favorable  results 
obtained  in  the  treatment  of  local  infections  by  artificially  produced 
passive  congestion. ^^  Too  severe  a  stasis,  with  resultant  edema,  prob- 
ably favors  local  infection.^" 

V.  Fodor^^  found  that  animals  surviving  infections  show  an  in- 
creased blood  alkalinity,  whereas  in  those  that  died,  the  alkalinity 
was  decreased;  also,  he  found  the  resistance  increased  b}^  intravenous 
injections  of  alkalies.  Other  observers^-  have  noted  a  decrease  in  re- 
sistance after  injecting  acids  into  the  blood.  According  to  Calabrese, 
the  alkalinity  of  the  blood  increases  in  immunization  of  animals 
against  toxins,  while  Cantani  found  the  injection  of  toxin  followed 
by  a  decrease  in  alkalinity.  Hamburger  has  shown  that  the  bac- 
tericidal power  of  the  blood  may  be  increased  in  vitro  bj^  shaking  it 
with  CO?  as  a  result  of  the  increased  alkalinity,  aided,  perhaps,  by 
some  slight  bactericidal  power  of  the  CO2  itself;  he  also  found  the 
blood  more  strongly  bactericidal  in  venous  congestion  than  normally, 
and  the  lymph  from  a  congested  part  was  also  found  more  strongly 
bactericidal  than  normal  lymph.  Hamburger^^  has  also  found,  how- 
ever, that  chemotaxis  is,  if  anything,  slightly  decreased  under  the  in- 
fluence of  CO2,  as  also  is  phagocytosis;  large  amounts  of  CO2  may 

^'Vierordt  (Arch.  f.  lleilk.,  1S7S  (19),  19:5)  found  coa{i;ul:ition  faster  in  the 
l)lood  in  passive  conj>;estion  than  in  normal  venous  blood;  but  Ihisebrock  (Zcit.  f. 
Biol.,  1882  (18),  41)  found  that  if  the  stasis  is  protracted,  the  coagulation  becomes 
delayed  because  of  the  excess  of  COa- 

•"*  Virchow's  Arch.,  1S99  (150),  329;  also.  "Osmotischcr  Druck  und  lonenlehre." 

"  Hee  Bier,  "IIypcr;emie  als  lleilmittel,"  Leipsic,  1903. 

'0  Glasewald,  C-ent.  Chenz.  Med.  Chir.,  1915  (IS),  507. 

f"  Cent.  f.  Bakt.,  1S90  (7),  753. 

•■^  Literature,  see  Ibunburf^er  (loc.  citJ'*),  p.  281 

'"•'  Virchow's  Arch.,  1899  (150),  329. 


THROMBOSIS  315 

reduce  the  phagocytic  power  for  coal  particles  by  2.5-50  per  cent. 
Hamburger's  results  as  to  the  bactericidal  power  of  human  blood  in 
venous  stasis  have  been  confirmed  by  Laqueur.'"*  Schiller  ascribes  this 
not  to  increased  alkalinity,  but  to  disintegration  of  leucocytes  with 
liberation  of  bactericidal  substances. ^^ 

The  blood  in  the  veins  and  capillaries  in  passive  congestion  is  gen- 
erally richer  in  corpuscles  than  normal,  perhaps  because  of  some  loss 
of  water,^*^  although  this  is  not  constant,  applying  particularly  to 
more  recent  or  more  local  processes;  in  long-continued  stasis,  as  in 
congenital  heart  disease,  the  blood  may  be  diluted."  In  the  concen- 
trated blood  of  passive  congestion  the  corpuscles  may  number  six  to 
eight  minions  per  cubic  millimeter,  while  the  concentration  of  the 
solids  of  the  serum  may  be  at  the  same  time  reduced  (Krehl).  The 
viscosity  of  such  blood  is  higher  than  that  of  normal  blood. ^^  In 
acute  stasis  the  proportion  of  serum  proteins,  especially  the  albumin, 
increases  with  the  duration  of  the  stasis;  no  changes  occur  in  the  non- 
protein constituents  of  the  blood  (Rowe).^^ 

THROMBOSIS 

The  chemistry  of  thrombosis  in  most  respects  resolves  itself  into  the 
chemistry  of  fibrin  formation,  a  subject  which  is  so  extensively  con- 
sidered in  most  treatises  on  physiological  chemistry  and  physiology 
that  it  does  not  seem  desirable  to  give  here  anything  more  than  the 
essential  principles  involved  in  the  clotting  of  the  blood,  as  now  under- 
stood, as  an  introduction  to  the  consideration  of  the  same  process  as 
it  occurs  under  pathological  conditions.  In  spite  of  innumerable  in- 
vestigations, our  knowledge  of  the  actual  participants  and  processes 
involved  in  the  formation  of  fibrin  is  in  a  very  unsatisfactory  and 
fragmentary  state.  Some  facts  seem  well  established,  however,  and 
we  have  a  general  idea  of  the  subject  that  may  be  applied  with  ad- 
vantage to  the  consideration  of  thrombosis. 

Fibrin  Formation^o 

Several  different  substances  seem  to  be  concerned  in  the  formation  of  fibrin, 
of  which  the  first  of  importance  is  its  antecedent,  fibrinogen.  Fibrinogen  is  a 
simple  protein,  related  to  the  globulins,  and  differing  chiefly  in  its  ready  coagula- 
bility, not  only  by  fibrin  ferment,  but  also  by  heat,  salts,  and  other  coagulating 
agencies.  By  itself,  however,  it  shows  no  tendency  to  coagulate  spontaneously. 
According  to  Goodpasture,"  fibrinogen  is  formed  through  the  combined  activity 

"  Zeit.  exp.  Path.  u.  Therap.,  1905  (1),  670. 

"  Beitr.  klin.  Chir.,  1913  (84),  H.  1. 

56  Grawitz,  Deut.  Arch.  f.  klin.  Med.,  1895  (54),  588. 

^' See  Krehl,  "  Pathologische  Physiologic,"  1904,  p.  201. 

58  Determann,  Zeit.  klin.  Med.,  1906  (59),  H.  2-4. 

"  Jour.  Lab.  Clin.  Med.,  1916  (1),  485. 

^°  For  literature  and  full  discussion  see  Hamniarsten's  or  Mathew's  Physiolog- 
ical Chemistry;  Morawitz,  Ergebnisse  der  Physiol.,  Abt.  1,  1904  (4),  307,  and 
Handbuch  d.  Biochem.,  1908  II  (2),  40;  Leo  Loeb,  Biochem.  Centr.,  1907  (6), 
829;  Howell,  Harvey  Lectures,  1917. 

"  Amer.  Jour.  Phvsiol.,  1914  (33),  70. 


316  DISTURBANCES  OF  CIRCULATION 

of  the  liver  and  intestines,  although  earlier  writers  have  variously  described  its 
formation  in  the  bone  marrow,  leucocytes,  liver  or  intestines.  The  amount  of 
fibrinogen  present  in  the  blood  is  actually  quite  small,  the  fibrin  formed  in  nor- 
mal clotting  being  but  0.1  to  0.4  per  cent,  of  the  weight  of  the  blood.  Acted 
upon  by  the  fibrin-ferment,  it  yields  the  characteristic  insoluble  protein  fibrin,  in 
crystalline  form  under  favorable  conditions,  ^^  but  we  do  not  know  definitely  what 
changes  the  fibrinogen  undergoes  in  this  process.  Fibrin  resembles  in  its  insolu- 
bility the  proteins  coagulated  by  heat,  alcohol,  etc.,  but  when  kept  aseptically  for 
some  time,  it  becomes  again  dissolved;  this  process  of  fibrinolysis  probably  de- 
pends upon  proteolytic  enzymes,  which  fibrin,  in  common  with  other  substances 
of  similar  physical  nature,  has  the  property  of  dragging  out  of  solution  and  holding 
firmly.  Undoubtedly  entangled  leucocytes  are  also  an  important  factor  in  the 
fibrinolysis,^'  which  is  greatly  increased  in  phosphorus  poisoning  and  when  the 
liver  is  excluded  from  the  circulation,  a  fact  suggesting  that  the  liver  may  form 
inhibiting  substances. 

Theories  of  Fibrin  Formation. — The  great  problem  is  the  nature  and  the  place 
and  manner  of  origin  of  the  fibrin-forming  enzyme,  generally  called  fibrin-jerment 
(also  plasmase,  thrombin  and  coagulin).  The  most  fundamental  theory  of  the 
origin  and  nature  of  fibrin-ferment  is  that  of  Alexander  Schmidt,  which  may  be 
briefly  described  as  follows:  The  ferment,  Schmidt  believed,  exists  in  the  plasma 
in  an  inactive  (prozyme  or  zymogen)  form,  which  he  called  prothrombin  Lpon 
disintegration  of  the  leucocytes  there  is  set  free  a  substance,  which,  acting  upon 
the  prothrombin,  converts  it  into  the  active  thrombin;  this  activating  agent 
Schmidt  designated  as  the  zymoplastic  substance.  With  various  modifications 
this  stands  to  the  present  day  as  a  basic  theory. 

It  having  been  shown  that  calcium  facilitates  the  formation  of  fibrin,  Pekel- 
haring  advanced  the  idea  that  the  prothrombin  does  not  exist  in  the  plasma,  but 
is  liberated  from  the  leucocytes,  and,  combining  with  the  calcium  of  the  plasma, 
forms  the  thrombin.  Morawitz  considers  three  substances  necessary  for  the 
formation  of  thrombin.  (1)  the  prothrombin  or  throjnbogen,  which  he  believes 
originates  in  the  blood-plates;  (2)  the  zymoplastic  stibstance  or  thrombokinase, 
which  is  liberated  from  the  leucocytes  into  the  plasma;  (3)  calcium  salts.  Howell,''* 
however,  explains  coagulation  as  follows:  Circulating  blood  normally  contains 
all  the  necessary  factors  for  fibrin  formation,  i.  e.,  fibrinogen,  prothrombin  and 
calcium.  But  there  is  also  present  an  inhibiting  substance,  antithrombin,^^  which 
prevents  the  calcium  from  activating  the  prothrombin  into  thrombin.  In  shed 
blood  there  appears  a  thromboplastin,  derived  from  the  platelets  or  the  tissues, 
which  neutralizes  the  antithrombin  and  thus  permits  thrombin  to  form.  Rett- 
ger^'^  holds  that  the  coagulation  of  the  blood  is  not  a  true  enzyme  action  at  all, 
while  Bordet  and  Delange*^  consider  that  thrombin  is  formed  by  the  interaction 
of  cytozyme  from  the  platelets  or  tissue  cells,  and  serozyme  of  the  plasma.  Mathews 
follows  Woolridge  and  considers  the  clotting  of  the  blood  as  essentially  the  crj^s- 
tallization  of  a  phospholipin-protein  compound,  blood  plasma,  the  stability  of 
which  compound  is  easily  upset  in  many  ways.  The  fibrin  threads  are  essen- 
tially liquid  crystals  coming  out  of  a  saturated  solution,  the  blood  plasma,  which 
is  practically  a  dilute  protoplasm.  It  will  not  serve  our  purpose,  however,  to  go 
further  into  the  hypotheses  and  disputes  over  these  questions,  which  are  detailed 
more  fully  in  the  literature  previously  cited,  but  it  may  be  stated  that  mimerous 
American  observers  have  found  Howell's  theory  to  fit  well  with  both  experimental 
and  clinical  observations  on  the  variations  in  the  coagulability  of  the  blood. 

The  question  has  been  raised  as  to  whether  the  leucocytes  or  platelets  secrete 
their  fibrin-forming  constituent  (be  it  thrombokinase  or  prothrombin  is  a  matter 

''^  See  Howell,  Amer.  Jour.  Physiol.,  1914  (35),  143;  Hekma,  Internat.  Zeit. 
physik.  chem.  Biol.,  1915  (2),  279. 

"'  Sec  Morawitz,  loc.  cit.;  also  Rulot,  Arch,  internat.  d.  Physiol.,  1904  (1),  152. 

8*  Amor.  .Jour.  Physiol,  1911  (29),  187. 

**  The  antithrombin  is  formecl  by  the  action  of  a  phosphatid  from  the  liver 
(heparin)  upon  a  pro-antithrombin  (Howell  and  Holt,  Amer.  Jour.  Phvsiol.,  191S 
(47),  328). 

««  Amer.  Jour.  Physiol.,  1909  (24),  40(). 

«^  Ann.  Inst.  Pasteur.,  1912  (20),  ()57.  See  also  Leo  and  Vincent,  Arch.  Int. 
Med.,  1914  (13),  398. 


COAGULATIOX  OF  HIJX)!)  317 

of  minor  importance  to  the  pathologist)  or  Uberate  it  only  after  their  disinte- 
gration. As  far  as  pathological  processes  go,  the  latter  seems  to  be  the  case,  the 
disintegration  ajjparently  occurring  whenever  tiie  leucocytes  come  in  contact  with 
a  foreign  body  or  with  dead  and  injured  tissues.  The  stroma  of  red  corpuscles 
also  contains  thrombokinase.""  Of  the  substances  that  may  be  isolated  from 
tissues,  ce-phalin  is  found  especially  active  in  producing  thrombosis,  and  may  be 
related  to  or  identical  with  the  thromboplastin.^" 

Tissue  Coagulins. — Among  the  other  i)oints  that  are  of  importance  in  i)atho- 
logical  conditions  is  the  fact  that  not  only  the  leucocytes,  but  also  tissue-cells, 
can  liberate  fibrin-forming  substances  {coaffidtiifi  is  the  non-committal  term  ap- 
plied by  Loeb).  Howell  considers  that  the  effect  of  the  tissue  "'coagulins"  is 
merely  to  neutralize  the  antithrombin  of  the  blood,  if  such  coagulins  actually 
e.Kist;  possibly  there  is  thromboplastin  in  the  tissues.  These  coagulating  agents 
are  present  in  tissue  extracts  and  are  liberated  whenever  the  tissues  are  injured; 
muscle  is  rich  in  coagulin,  as  are  also  the  liver  and  kidney,  and,  which  is  par- 
ticularly important,  the  blood-vessel  wall  (L.  Loeb).  Pieces  of  these  tissues 
})laced  in  contact  with  fibrinogen  solution  will  bring  about  prompt  clotting.  An- 
other important  fact  is  that  the  coagulins,  whether  derived  from  leucocytes  or 
from  the  tissues,  have  a  certain  degree  of  sjiecificity — that  is,  they  act  solely  or 
most  rapidly  with  fibrinogen  of  blood  of  the  species  from  which  they  are  obtained.'" 
In  some  instances  this  specificity  is  absolute,  but  more  generally  (particularly  in 
the  mammalia)  it  is  only  relative.  Loeb  also  found  that  the  amount  of  tissue 
coagulin  was  not  decreased  in  organs  altered  by  phosphorus  poisoning,  although 
during  experimental  autolysis  the  coagulins  disappear.  Wlien  tissue  coagulins 
and  blood  coagulins  act  together,  the  effect  is  greater  than  the  sum  of  their  inde- 
pendent actions,  indicating  the  probability  that  they  combine  in  some  way  to 
produce  a  particularly  active  coagulin.  The  blood  coagulins  are  quite  different 
from  the  tissue  coagulins  in  many  important  respects,  and  the  coagulins  cannot 
be  looked  upon  as  a  single  substance  of  different  origins. 

Blood-platelets. — It  is  still  undetermined  just  what  part  the  platelets  play  in 
coagulation.  The  well-known  observation  that  in  thrombosis  the  fibrin  is  often 
first  formed  about  masses  of  platelets  clinging  to  the  wall  of  the  vessel  indicates 
that  they  participate  in  the  process,  and  Bizzozero  and  others  have  maintained 
that  the  platelets  and  not  the  leucocytes  are  the  source  of  the  prothrombin. 
Numerous  studies  on  the  relation  of  the  platelets  to  disease  conditions  have  in- 
dicated a  certain  parallelism  between  their  number  and  the  tendency  to  coagula- 
tion observed  in  the  various  diseases  (Welch).  Howell  believes  the  platelets  to  be 
the  chief  source  of  thromboplastin,  which  neutralizes  the  antithrombin  of  the 
blood  and  thus  causes  clotting.  Wright  and  Minof^  find  that  a  viscous  meta- 
morphosis of  the  platelets  is  intimately  associated  with  the  early  stages  of  coagu- 
lation. Bordet  and  Delange  consider  the  platelets  of  more  importance  than  the 
leucocytes  in  producing  participants  of  the  coagulating  mechanism.  The  histo- 
logical evidence  of  the  importance  of  the  platelets  in  thrombus  formation  is  con- 
clusive (see  Zurhelle,  Derewenko),  and  Cramer  and  Pringle"^  state  that  coagulation 
cannot  occur  without  platelets.  However,  the  blood  of  fishes,  birds  and  reptiles 
clots  although  no  platelets  are  found  in  these  animals.  Human  thoracic  lymph 
also  is  devoid  of  platelets  yet  it  clots;  but  it  may  contain  products  of  platelet 
disintegration  to  explain  this  clotting  (Jordan)."'*  Kemp'*  concludes,  from  a 
thorough  review  of  the  subject,  that  the  blood-platelets  are  usually  normal  or 
subnormal  in  number  during  acute  infectious  diseases,  but  increase  rapidly  if  the 
disease  terminates  by  crisis;  iii  pernicious  anemia  the  number  is  always  greatly 
diminished,  although  in  secondary  anemias  they  may  sometimes  be  increased;  in 
purpura  hemorrhagica  the  number  of  plates  is  enormously  diminished,  which  is 

««  Barratt,  Jour.  Path,  and  Bact.,  1913  (17),  303. 

"Howell,  Amer.  Jour.  Physiol.,  1912  (31),  1;  MacLean,  ibid.,  1916  (41),  250; 
1917  (43),  586. 

'"Leo  Loeb,  Univ.  of  Penn.  Med.  Bull.,  1904  (16),  382;  Muraschew,  Deut. 
Arch.  klin.  Med.,  1904  (SO),  187. 

"  Jour.  Exp.  Med.,  1917  (26),  395. 

"  Quart.  Jour.  Exper.  Physiol,  1913  (6),  1. 

"  Anat.  Rec,  1918  (15),  37. 

""  Jour.  AmQv.  Med.  Assoc,  1906  (46),  1022. 


318  DISTURBANCES  OF  CIRCULATION 

perhaps  related  to  the  slowness  of  the  clotting  of  the  blood  in  this  condition. 
Duke"  states  that  when  the  platelet  count  falls  below  10,000  per  cubic  mm.  there 
is  delayed  coagulation  and  a  tendency  to  purpura;  with  counts  above  40,000 
there  is  usually  no  hemorrhagic  tendency.""  If  the  platelet  count  is  reduced  arti- 
ficially (by  benzene,  diphtheria  toxin)  a  similar  purpuric  tendency  is  observed. 
Poisons  that  in  large  doses  reduce  the  platelet  count,  will  increase  it  if  in  small 
doses. 

Calcium  Salts. — The  exact  significance  of  calcium  in  fibrin  formation  is  still 
unsettled.  Blood  from  which  the  calcium  has  been  precipitated  will  not  coagu- 
late, and  the  addition  of  calcium  salts  will  promptly  cause  it  to  do  so.  The  vari- 
ous hypotheses  advanced  to  explain  the  way  in  which  calcium  influences  the 
clotting  process  are  not  in  agreement.  One  hypothesis  is  that  the  calcium  ions 
are  necessary  for  the  transformation  of  prothrombin  into  thrombin  (Pekelharing, 
Hammarsten,  Morawitz),  the  thrombin  consisting  of  a  compound  of  prothrombin, 
calcium  salts,  and  thrombokinase.  Howell  considers  that  no  kinase  is  necessary, 
the  calcium  activating  the  prothrombin  whenever  it  is  not  inhibited  by  anti- 
thrombin. 

Modification  of  Coagulability. — If  blood  is  drawn  into  a  glass 
vessel  well  coated  with  oil  or  vaseline,  through  a  cannula  similarlj^  pro- 
tected, no  coagulation  will  take  place;  but  if  any  unoiled  foreign  sub- 
stance enters,  even  particles  of  dust,  coagulation  begins  at  once.  The 
explanation  is  that  the  leucocj^tes  do  not  liberate  their  coagulating 
substances  until  they  have  been  injured  by  contact  with  some  foreign 
body,  and  the  experiment  proves  the  importance  of  this  action  of  the 
leucocytes,  as  well  as  explaining  why  the  blood  does  not  coagulate  dur- 
ing life.  The  classical  experiment  of  the  ligation  of  a  vein  without 
injury  to  the  endothelium,  which  permits  the  blood  to  remain  stag- 
nant for  a  long  period  without  clotting,  depends  upon  the  same  fact, 
namely,  that  normal  endothehum  neither  liberates  coagulin  itself  nor 
injures  the  leucocytes  so  that  they  disintegrate.  Loeb  recalls  the 
observation  of  Overton  that  lipoids  are  important  constituents  of  the 
cell  membranes,  and  suggests  a  similarity  between  the  vessel  lining 
and  the  oiled  cannula,  but  analyses  of  aortic  endothelium  have  shown 
a  rather  low  lipin  content  (8.41-9.25  per  cent.),  although  peritoneal 
endothehum  has  much  more  (13  to  15  per  cent.).'''^  The  suggestion 
that  the  vessel  walls  contain  an  anti-coaguUn  could  not  be  confirmed  by 
Loeb.  Since  leucocytes  are  constantly  undergoing  disintegration  in 
the  blood  and  tissues  under  normal  conditions,  it  might  be  asked  why 
they  do  not  cause  clotting  then  and  there.  In  explanation  Loeb  ad- 
vances his  observation  that  the  coagulins  are  destroyed  during  cell 
autolysis,  and  suggests  that  when  leucocytes  normally  thsintcgrate,  the 
coagulins  are  first  destroyed  by  autolysis.  It  has  also  been  shown  that 
the  cells  and  serum  contain  substances  which  inhibit  or  prevent 
coagulation,  and  it  is  possible  that  these  play  an  important  part  under 
normal    conditions   in    preventing   coagulation    by   products   of  cell 

"Jour.  Exp.  Med.,  1911  (14),  265;  Arch.  Int.  Med.,  1912  (10),  445;  Jour. 
Amer.  Med.  Assoc,  1915  (65),  1600. 

""Gram  (Arch.  Int.  Med.,  1920  (25),  325)  states  that  platelet  counts  below 
100,000  goncrallv  accompany  a  tendency  to  bleed.  He  gives  the  normal  figure  as 
200,000  to  500,000,  but  usually  over  300,000. 

"Tait,  (iuart.  Jour.  Exp.  Physiol.,  1915  (S),  391. 


COAGULATION  OF  BLOOD  319 

disintegration,  nnicli  as  other  antienzymes  are  supposed  to  act  in 
prevontins  autodigcstion  of  living  cellp. 

Coagulation  of  drawn  blood  may  be  retarded  experimentally  by  re- 
moval of  the  calcium  by  precipitation  as  oxalate,  fluoride,  etc.;  also  by 
diminishing  the  oxygen  and  increasing  the  COi,,  by  addition  of  solu- 
tions of  neutral  salts  in  large  amounts,  by  diluting  greatly  with  water, 
or  by  keeping  the  blood  cold.  Bile  salts  retard  coagulation  markedly, 
by  interfering  with  the  conversion  of  fibrinogen  into  fibrin."  Coagu- 
lation may  be  hastened  by  moderate  heat,  by  whipping,  exposure  to 
air,  by  contact  with  much  foreign  matter,  and  by  the  addition  of 
watery  extracts  from  many  different  tissues  and  organs.  Poisons  that 
destroy  the  platelets  reduce  the  coagulation  (Duke).  Of  particular 
interest  pathologically  is  the  retardation  of  coagulation  that  follows 
injections  of  proteoses  (the  so-called  "peptone"  solutions)  and  also 
various  other  protein-containing  solutions,  such  as  organ  extracts, 
bacterial  toxins,  snake  venoms,  eel  serum,  extract  of  leeches  or  of 
Uncinaria,  impure  nucleo-protein  solutions,  or  solutions  of  various 
colloids.  Most  of  these  substances  e.  g.,  peptone,  eel  serum,  cause 
reduction  of  coagulability  when  injected  into  animals,  and  are  without 
effect  on  blood  removed  from  the  body.  A  few,  however,  prevent 
coagulation  of  drawn  blood  (snake  venom,  extract  of  leeches).  When 
substances  of  the  first  class  are  injected  in  sufficient  quantities,  there 
occurs  first  a  period  of  accelerated  coagulation  which  may,  particularly 
in  the  case  of  organ  extracts,  cause  prompt  death  from  intravascular 
clotting;  if  the  animal  survives,  there  follows  a  period  of  decrease 
or  total  inhibition  of  coagulability  of  the  blood,  both  within  the  ves- 
sels and  after  removal  from  the  body.  The  first  period  of  increased 
coagulability  undoubtedly  depends  upon  the  formation  of  a  large 
amount  of  fibrin-ferment,  but  it  has  not  yet  been  satisfactorily  ex- 
plained how  the  inhibition  of  coagulation  is  produced.  Apparently 
the  fibrin-ferment  formed  at  first  is  rapidly  destroyed,  but  it  is  thought 
by  some  that  it  is  converted  into  a  substance  that  neutralizes  the 
fibrin-ferment  that  may  be  formed  later,  or  that  a  true  anticoagulin 
is  formed.  It  is  also  among  the  possibilities  that  all  the  available 
prothrombin  or  thrombokinase  is  used  up  during  the  first  stage  of 
acceleration.  As  before  mentioned,  the  blood  and  tissues  contain 
substances  that  inhibit  coagulation,  and  it  may  be  that  these  are 
secreted  in  excessive  amounts,  a  view  which  is  receiving  much  support 
from  recent  observations.  According  to  Davis""  injection  of  tlirombin 
is  followed  quickly  by  an  increase  in  the  amount  of  antithrombin  in  the 
blood.  It  has  been  found  that  in  animals  deprived  of  the  liver  no 
coagulation-inhibiting  substances  are  formed  in  the  blood  after  in- 
jection of  proteoses,  hence  Delezenne  believes  that  the  substances  of 
this  class  act  by  causing  a  destruction  of  leucocytes,  thus  hberating  a 

"  Haessler  and  Stebbins,  Jour.  Exp.  Med.,  1919  (29),  445. 
"«Amer.  Jour.  Physiol.,  1911  (29),  IGO. 


320  DISTURBANCES  OF  CIRCULATION  . 

substance  which  increases  coagulation  and  also  another  substance 
retarding  coagulation;  the  first  of  these  is  destroyed  by  the  liver,  leaving 
the  retarding  substance  to  act  unopposed.^*  Leech  extract  (hirudin) 
prevents  clotting  by  means  of  an  antiferment  action,  combining  with 
the  thrombin. ^^  Snake  venom,  however,  acts  upon  the  thrombokinase 
(Morawitz). 

Coagulability  of  the  Blood  in  Disease. — In  disease  the  altera- 
tions in  the  coagulability  of  the  blood  depend  upon  much  the  same 
factors.  In  all  conditions  associated  with  suppuration  and  leucocyto- 
sis  the  amount  of  fibrinogen  is  increased.  This  is  especially  true  of 
pneumonia.^°  The  fluidity  of  the  blood  in  septicemia  is  probably 
dependent  upon  the  appearance  of  the  coagulation-inhibiting  phase 
that  follows  the  action  of  the  products  of  cell  destruction,  including 
among  them  proteoses.  In  this  connection  should  be  mentioned  the 
observation  of  Conradi,^^  who  found  that  among  the  products  of  au- 
tolysis is  a  coagulation-inhibiting  substance  which  is  not  destroyed  by 
heat,  diffuses  readily,  and  in  general  behaves  unlike  the  proteins.  This 
or  similar  substances  may  well  play  a  part  in  affecting  coagulation  in 
infectious  diseases,  and  Whipple^^  has  found  a  decreased  coagula- 
bility in  septicemia  because  of  the  presence  of  an  excess  of  anti- 
thrombin.  It  may  also  be  mentioned  that  animals  soon  acquire  an 
immunity  against  proteoses,  so  that  their  inhibiting  influence  is  no 
longer  shown.  This  corresponds  to  the  observation  of  Kanthack^^ 
that  immune  serum  against  venom  neutralizes  very  effectively  the 
anticoagulating  principle  of  venom;  an  amount  of  antiserum  alto- 
gether insufficient  to  neutralize  the  toxic  properties  of  venom  will 
neutralize  its  property  of  preventing  clotting.  The  bacterial  prod- 
ucts may  also  modify  coagulation,  and  L.  Loeb^"*  has  found  that 
different  organisms  are  unequally  effective  in  this  respect,  Staphylo- 
coccus aureus  being  much  more  powerful  in  causing  coagulation  than 
any  others  tested ;^^  typhoid,  diphtheria,  tubercle,  and  xerosis  bacilli 
and  streptococci  being  without  any  apparent  effect,  while  pyocyan- 
eus,  prodigiosus,  and  colon  bacilli  occupy  an  intermediate  position. 
Furthermore,  after  the  organisms  are  killed  bj-  boiling,  this  effect  is 
greatly  reduced,  showing  that  it  does  not  depend  merely  upon  the 
mechanical  action  of  the  bacteria,  but  probabi}'  upon  bacterial  prod- 
ucts contained  in  the  culture-media. 

^*  Tlic  manner  in  which  gelatin  injections  afl'ect  the  blooil  coagulability  is  not 
yet  understood  (see  Hoggs,  Deut.  Arch.  klin.  Med..  1<)04  (79),  539);  Moll  (Wien. 
klin.  Woch.,  1903  (16),  1215)  found  an  increase  in  fibrinogen. 

"  Hirudin  may  contain  antikinase  (Mellanbv,  Jour,  of  I'hvsiol.,  1909  (38),  441). 

8"  Dochez,  Jour.  Exp.  Med.,  1912  (10),  093. 

"  Hofmeister's  Beitr.,  1901  (1),  137. 

s'' Arch.  Int.  Med.,  1912  (9),  305. 

'*  Cited  1)V  Lazarus-Barlow,  p.  141. 

«<  Jour.  Aicd.  Research,  1903  (10),  407. 

*^  Much  (Biochem.  Zeit.,  1908  (14),  143)  states  that  staphylococcus  contains 
thrombokinase. 


COAGL'LATIOX  OF  HLOOI)  321 

After  phosphorus  poisoning  the  blood  may  become  non-coagula- 
ble,  whicli  Jacoby*^  ascribed  to  an  absence  of  fibrinogen  in  the  blood, 
because  of  a  fibrinogen-destroying  ferment  in  the  liver.  Doyon^^  has 
made  a  similar  finding  in  chloroform  necrosis  of  the  liver,  but  he  at- 
tributes especial  importance  to  an  excess  of  antithrombin  liberated 
from  the  liver  in  these  conditions.  Whipple  has  also  found  a  de- 
crease in  fibrinogen  with  chloroform  necrosis  and  cirrhosis  of  the 
liver. ^^  In  other  instances  of  decreased  coagulability  the  fibrinogen 
is  present,  generally  in  normal  amounts.  After  death  the  blood  be- 
comes incoagulable  because  the  fibrinogen  is  destroyed  through  a 
process  similar  to  that  of  fibrinolysis;*^  this  fibrinolysis  may  be  com- 
plete as  early  as  ten  hours  after  death.  The  other  proteins  of  the 
blood  do  not  seem  to  be  correspondingly  attacked.  Thrombokinase 
is  also  scanty  in  cadaver  blood,  but  there  seem  to  be  no  coagulation- 
inhibiting  substances  present.  In  anaphylactic  shock  the  coagula- 
bility is  reduced  or  abolished,  associated  with  which  is  a  leucopenia.^° 

Whipple^^  states  that  the  antithrombin-prothrombin  balance  in  the 
blood  is  in  delicate  equilibrium,  but  preserved  by  strong  factors  of 
safety.  The  prothrombin  factor  is  rarely  involved,  most  notably  in 
melena  neonatorum  and  aplastic  anemia,  and  such  conditions  may  be 
relieved  by  injecting  normal  blood,  through  the  added  prothrombin. 
The  antithrombin  factor  is  often  excessive  in  hemorrhagic  conditions, 
especially  with  hepatic  injury,  or  it  may  be  lowered  and  lead  to  throm- 
bosis from  relatively  slight  injuries.  Obviously  the  injection  of  nor- 
mal blood  will  harm  rather  than  help  patients  with  hemorrhage  due 
to  excessive  antithrombin.  Antithrombin  is  often  found  increased  in 
diseases  of  the  blood-forming  organs,  e.  g.,  leukemia,  possibly  as  a 
reaction  to  the  products  of  disintegration  of  corpuscles;  and  hence 
hemorrhagic  tendencies  are  noted  in  these  diseases.  In  icterus  the 
notable  tendency  to  hemorrhage  seems  to  depend  upon  the  binding  of 
the  calcium  of  the  blood. by  the  bile  pigments,^^  and  administration 
of  calcium  may  bring  the  coagulation  time  back  to  normal  with  a  cor- 
responding decrease  in  the  hemorrhagic  tendency. 

Pfeiffer^^  estimated  the  fibrin  content  of  the- blood  in  disease,  and 
found  it  increased  in  diseases  with  leucocytosis  (pneumonia,  rheuma- 
tism, erysipelas,  scarlet  fever),  except  leukemia,  where  it  was  normal; 

"Zeit.  physiol.  Chem.,  1900  (30),  175;  also  Doyon  et  al,  Compt.  Rend.  Soc. 
Biol.,  1905  (58),  493. 

"Compt.  Rend.  Soc.  Biol.,  1905  (58),  704;  Jour.  phys.  et  path.,  1912  (14), 
229. 

«8  Bull.  Johns  Hopkins  Hosp.,  1913  (24),  207. 

8'  Morawitz,  Hofmeister's  Beitr.,  1906  (8),  1. 

^°  The  incoagulability  of  menstrual  blood  is  ascribed  to  a  lack  of  fibrin  ferment 
by  Bell  (Jour.  Path,  and  Bact.,  1914  (18),  462)  and  to  an  excess  of  antithrombin 
by  Dienst  (Miinch.  med.  Woch.,  1912  (51),  2799). 

"1  Arch.  Int.  Med.,  1913  (12),  637. 

"  Lee  and  Vincent,  Arch.  Int.  Med.,  1915  (16),  59. 

»3  Zeit.  klin.  Med.,  1897  (33),  214;  Cent.  f.  inn.  Med.,  1898  (19),  1. 

21 


322  DISTURBANCES  OF  CIRCULATION 

in  diseases  without  leucocytosis  (typhoid,  malaria,  nephritis),  the 
fibrin  was  normal  in  amount.  Stassano  and  Billon^^  have,  further- 
more, shown  that  the  amount  of  fibrin-ferment  varies  directlj^  with 
the  number  of  leucocytes  in  the  blood.  Kollmann^^  found  an  increase 
in  the  fibrin  of  eclampsia,  which  Lewinski^^  could  not  substantiate. 
In  experimental  infections  of  anitnals  Langstein  and  Mayer^"  found 
a  specific  increase  in  pneumococcus  sepsis,  which  undoubtedly  bears 
an  important  relation  both  to  the  characteristic  fibrinous  nature  of 
the  alveolar  exudate  in  pneumonia,  and  the  striking  amount  of  fibrin 
found  in  pneumococcus  pleuritis,  peritonitis,  etc.  Mathews^^  found 
an  increase  in  the  fibrin  with  all  experimental  suppurations. 

The  coagulation  time  determined  by  different  methods,  in  which 
different  conditions  for  coagulation  are  presented,  varies  from  2  to  30 
minutes;  with  most  methods  it  is  5  to  8  minutes. ^^  In  general, 
coagulability  is  not  constantly  if  at  all  altered  bj^  fever,  cancer,  dia- 
betes, slight  secondary  anemias,  or  many  other  diseases,  and  in  nor- 
mal conditions  it  remains  fairly  constant.  In  infants  the  coagulation 
time  is  slightly  shorter  than  in  adults.  The  coagulation  is  hastened 
after  considerable  hemorrhages,  in  endocarditis,  and  perhaps  in  aneu- 
rism and  thrombosis;  and  is  commonly  delayed  in  the  acute  exan- 
themata, in  hemophilia,  in  purpura  neonatorum,  and  occasionally  in 
some  other  diseases.^  There  is  entire  lack  of  agreement  concerning 
the  reputed  acceleration  of  coagulation  by  oral  administration  of  cal- 
cium salts,  and  retardation  by  citrates;  and  the  supposed  thrombo- 
plastic  influence  of  gelatin  cannot  be  shown  consistently  by  direct 
observations.  In  jaundice,  calcium  salts  probably  have  an  effect,  since 
here  the  cause  of  the  deficient  coagulation  seems  to  be  the  fixation  or 
precipitation  of  the  blood  calcium  by  the  bile  pigments.  The  bile 
salts  also  prevent  the  conversion  of  fibrinogen  into  fibrin.''^  It  seems 
probable  that  the  measurement  of  the  time  required  for  coagulation  to 
take  place  in  vitro  does  not  exactly  represent  the  tendency  of  the  same 
blood  to  coagulate  in  the  body  of  the  person  from  whom  it  is  obtained; 
for  example,  the  injection  of  foreign  serum  has  a  notable  effect  in  stop- 
ping hemorrhages,  but  the  coagulation  time  of  the  recipient's  blood  is 
not  correspondingly  altered.  Whipple's  observation  that  with  a  low 
fibrinogen  content  the  blood  may  coagulate  in  normal  time,  and  yet  the 
clots  be  too  delicate  to  stop  hemorrhage,  explains  at  least  part  of  the 
discrepancy;  and  of  similar  signifiance  is  the  fact  that  with  a  very 

"  Compt.  Rend.  See.  Biol.,  1903  (55),  511. 

»5Cent.  f.  Gyniik.,  1897  (21),  341. 

»«  Pfliiger's  Arch.,  1903  (100),  (ill. 

"  Hofnieister's  Bcitr.,  1903  (5),  09. 

»«  Amer.  Jour.  Physiol.,  1899  (3),  53. 

*"  Full  review  and  bibliography  by  Cohen,  Arch.  Int.  Med.,  1911  (8),  084  and 
820. 

'  See  Dochez  (Jour.  Exp.  Med.,  1912  (10),  093),  who  found  some  delay  in 
coagulation  in  pneumonia.  Corroborated.by  Minot  and  Lee,  Jour.  Anier.  Med. 
Assoc,  1917  (08),  645. 


THROMBOSIS  323 

low  platelet  count  the  blood  may  coaguhite  as  rapidly  as  normal,  but 
the  clots  do  not  shrink  and  become  firm  (Duke).  Hence  with  a  se- 
vere purpura  hemorrhagica  wc  may  have  a  normal  clotting  time. 
In  other  conditions  with  normal  coagulability,  hemorrhages  may  re- 
sult from  excessive  fibrinolysis  which  causes  solution  ol"  the  clot, espe- 
cially in  hepatic  diseases.  ^ 

The  Formation  of  Thrombi 

If  we  apply  the  facts  brought  out  in  the  preceding  discussion  rela- 
tive to  the  factors  in  the  coagulation  of  blood,  to  the  manner  and 
conditions  under  which  thrombi  are  formed  in  the  circulating  blood, 
we  find  explanations  for  many  of  the  features  of  thrombosis.  Welch^ 
describes  the  steps  in  the  formation  of  a  thrombus  after  injury  to  the 
vessel-wall,  as  follows:  First,  there  is  an  accumulation  of  blood- 
platelets  adhering  to  the  wall  at  the  point  of  injury.  Leucocytes, 
which  may  be  present  in  small  numbers  at  the  beginning,  rapidly  in- 
crease in  number,  collecting  at  the  margins  of  the  platelet  masses  and 
between  them.  Not  until  the  leucocytes  have  accumulated  does  the 
fibrin  appear.  As  Welch  remarks,  these  findings  afford  no  conclusive 
evidence  as  to  whether  fibrin-ferment  is  formed  from  the  leucocj'tes 
or  from  the  platelets,  but  since  the  fibrin  does  not  appear  until  after 
the  leucocytes  have  accumulated,  and  also  since  small  thrombi  may 
consist  solely  of  platelets  without  fibrin,  it  seems  probable  that  the 
leucocytes  must  be  looked  upon  as  the  chief  source  of  the  ferment. 
If  the  blood  is  made  incoagulable  by  injection  of  hirudin,  injury  to 
the  vessel-walls  causes  the  formation  of  thrombi  composed  entirely  of 
platelets  (Schwalbe).  Sometimes  small  clots  may  form  without  the 
apparent  participation  of  either  platelets  or  leucocytes.  These  purely 
fibrinous  thrombi  seem  to  start  from  injured  endothelial  cells,  par- 
ticularly in  inflammatory  conditions,  such  as  pneumonic  lungs,  and 
give  the  impression  that  the  coagulin  is  derived  from  the  endothelial 
cells.  Zurhelle  attributes  by  far  the  most  important  part  to  the 
platelets,  an  opinion  shared  by  many,  including  Derewenko,'*  who 
holds  that  the  coagulation  of'  blood  with  entirelj^  occluded  vessels  is 
quite  distinct  from  true  thrombosis  because  of  the  lack  of  platelets 
in  stagnant  blood. ^  Clots  formed  in  the  absence  of  platelets  do  not 
shrink  like  proper  thrombi  (Duke). 

The  process  of  clotting  in  the  stoppage  of  hemorrhage  offers  some 

2  See  Goodpasture,  Bull.  Johns  Hopkins  Hosp.,  1914  (25),  330. 

^  Albutt's  System,  vol.  6,  complete  discussion  of  the  general  features  of  throm- 
bosis; also  see  Kiister,  Ergeb.  inn.  Med.,  1913  (12),  667;  Zurhelle,  Ziegler's  Beitrage, 
1910  (47),  539;  Schwalbe,  Ergebnisse  Pathol.,  1907  (XI  (2)  )  901;  Lubarsch, 
AUg.  Pathol.,  Vol.  1,  Wiesbaden.  1905.  See  Aschoff,  Ziegler's  Beitr.,  1912  (52), 
205,  and  Arch.  Int.  Med.,  1913  (12),  503,  concerning  the  mechanics  of  thrombus 
formation. 

*  Ziegler's  Beitr.,  1910  (48),  123. 

^  Not  accepted  by  Schwalbe,  loc.  cit.^ 


32-1  DISTURBANCES  OF  CIRCULATION 

differences  from  intravascular  clotting,  in  that  the  coagulins  of  the 
tissue-cells  also  come  into  play.  It  is  rather  difficult  to  determine 
how  much  of  the  coagulation  depends  on  these,  and  how  much  on  the 
coagulins  of  the  leucocytes,  for  the  same  conditions  that  favor  libera- 
tion of  tissue  coagulins,  i.  e.,  much  laceration  and  destruction  of  the 
tissue,  also  favor  the  disintegration  of  leucocytes  by  offering  large 
^reas  of  surface  for  contact.  Loeb  is  of  the  opinion,  however,  that 
-of  the  two,  the  latter  factor  is  the  more  important.  It  may  be  re- 
called that  the  joint  action  of  tissue  and  blood  coagulins  is  greater 
than  the  sum  of  their  individual  actions,  which  also  must  be  an  im- 
portant factor  in  causing  clotting  in  bleeding  wounds. 

As  to  the  relative  importance  of  stagnation  and  vessel  injury  in 
producing  thrombosis,  we  know  that  total  stasis  in  an  uninjured  vessel 
may  not  result  in  thrombosis,  and,  on  the  other  hand,  extensive  in- 
jury or  large  calcified  plaques  in  the  intima  of  the  aorta  may  also 
cause  no  thrombosis  because  of  the  rapidity  of  the  blood  flow;  and, 
furthermore,  clotting  may  occur  even  in  intact  vessels  under  the  influ- 
ence of  substances  liberating  fibrin-ferment  in  the  blood;  e.  g.,  snake 
venoms,  nucleoprotein  injections,  and  possibly  in  disease.  As  the  red 
corpuscles  contain  thromboplastic  substances  we  may  have  thrombi 
formed  when  hemolytic  agents  are  present  in  relatively  stagnant 
blood,  even  without  injury  to  the  vessel-walls.^  Presumably  the  clot- 
ting does  not  occur  when  the  stream  is  rapid,  because  any  fibrin- 
ferment  that  may  be  liberated  by  injured  leucocytes  or  endothelium 
is  swept  away  before  fibrin  can  become  attached  to  the  vessel-wall; 
or,  according  to  Howell's  hypothesis,  because  the  current  brings  an 
excess  of  antithrombin  to  the  point  where  the  thromboplastin  is  being 
formed.  Naturally,  the  combination  of  an  injured  vessel-wall,  a  slow 
current,  and  a  high  coagulability  offer  the  most  favorable  conditions, 
and  we  owe  to  Welch  the  appreciation  of  the  fact  that  in  a  large  pro- 
rportion  of  all  thrombi,  even  those  caused  by  apparently  purely  me- 
rchanical  agencies  (e.  g.,  cardiac  incompetence),  bacteria  are  present 
and  probably  determine  the  injury  to  the  vessel-walls  and  the  libera- 
tion of  fibrin-ferment.^  We  have  previously  referred  to  L.  Loeb's 
.  observations  on  the  effect  of  bacteria  in  causing  coagulation  of  the 
blood. 

Hyalin  thrombi  are  frequently  the  cause  of  extensive  degenerative  lesions  in  the 
viscera,  and  although  commonly  formed  of  red  corpuscles,  they  do  not  stain  at  all 
like  normal  corpuscles,  presumably  because  a  certain  proportion  of  the  hemoglobin 
has  been  altered  or  lost  through  hemolysis.  Of  particular  interest  is  their  reaction 
to  Weigert's  fibrin  stain,  by  which  they  often,  but  not  always,  stain  intensely, 
especially  when  hardened  in  Zenker's  solution;  a  fact  that  has  been  the  cause  of 
much  confusion  in  earlier  studies.  Flexner*  first  appreciated  the  nature  of  these 
thrombi    as    originating    from    agglutinated    red    corpuscles,   although   Hobs, 

6  Dietrich  Cent.  f.  Path.  (Verhandl.),  1912  (23),  372. 

^  Welch,  Venous  Thrombosis  in  Cardiac  Disease,  Trans.  Assoc.  Amer.  Phys., 
1900,  vol.  15. 

8  Jour.  Med.  Research,  1902  (8),  316. 


EMBOLISM  325 

Ziegler,  and  others  had  earlier  suggested  that  hyalin  thrombi^were  formed  from 
red  corpuscles.  Boxnieyer'  independently  arrived  at  the  same  conclusion  as 
Flexner,  in  studying  hyalin  thrombi  as  the  cause  of  necrosis  in  the  liver  of  animals 
infected  with  the  hog-cholera  bacillus.  Flexner  produced  hyalin  thrombi  by 
injecting  corpuscles  agglutinated  by  ricin,  or  by  injecting  ricin  itself,  or  hemolytic 
substances  such  as  ether  or  foreign  serum.  As  the  thrombi  become  old,  the  cor- 
l)uscles  lose  their  form  and  color  and  produce  the  typical  hyalin  appearance. 
iVarce'"  proved  conclusively  the  dependence  of  the  thrombus  formation  upon 
agglutination,  for  he  secured  the  same  results,  including  the  liver  necrosis,  by 
injecting  specific  agglutinating  serums.  He  states  that  fibrin  threads  may  oc- 
casionally be  found  at  the  periphery  of  the  larger  thrombi,  but  never  in  the  smaller 
ones.  It  is  extremely  probable,  from  Flexner's  observations,  that  in  the  thrombosis 
produced  by  injecting  various  toxic  substances  into  the  blood,  the  so-called  "fibrin 
ferment  thro7nbosis,"  the  thrombi  are  merely  agglutinative  thrombi,  devoid  of 
fibrin;  this  is  undoubtedly  true  for  many  of  the  thrombi  observed  after  poisoning 
with  the  powerfully  agglutinative  snake  venoms  (see  Chap.  vi).  Bacterial  hemag- 
glutinins may  also  cause  the  formation  of  hyalin  thrombi.'^  On  the  other  hand, 
some,  at  least,  of  the  hyalin  capillary  thrombi  are  undoubtedly  composed  of  soft 
masses  of  fibrin  which  have  not  become  fibrillar,  although  the  successful  staining 
by  fibrin  stain  is  not  final  proof  of  the  fibrinous  nature  of  a  thrombus.  The  liver 
necrosis  which  follows  ether  injections  in  animals  is  caused  by  fibrinous  thrombi 
which  result  from  liberation  of  coagulins  by  the  injured  cells  (L.  Loeb ). 

Secondary  Changes  in  Thrombi. — The  changes  that  occur  in  thrombi  after 
they  have  existed  for  some  time  are  largely  due  either  to  ingrowth  of  new  tissue  or 
to  calcification,  the  latter  of  which  will  be  considered  in  a  separate  chapter  The 
only  other  change  of  interest  from  the  chemical  standpoint  is  the  central  softening 
which  may  occur  in  any  large  thrombus,  but  is  particularly  often  observed  in  the 
large  globular  thrombi'found  in  the  heart.  The  center  of  the  thrombus  may  be 
so  completely  softened  that  it  resembles  a  sac  of  pus,  the  contents,  according  to 
Welch,  consisting  of  necrotic  fatty  leucocytes,  albuminous  and  fatty  granules, 
blood-pigment  and  altered  red  corpuscles,  and  occasionally  acicular  cr^'stals  of 
fatty  acids.  Undoubtedly  this  softening  is  related  to  the  process  of  fibrinolysis 
previously  described,  and  depends  upon  digestion  of  the  fibrin  by  leucocytic 
enzymes,^^  but  the  fact  that  the  central  portion  alone  undergoes  softening  is  of 
interest,  suggesting  that  the  antibodies  for  leucocytic  proteases,  which  Opie^' 
found  present  in  normal  serum,  prevent  digestion  at  the  surface  of  the  clot.  The 
same  fact  indicates  that  the  tissue  fibrinolysins^^  do  not  play  an  active  part  in 
softening  clots. 

Embolism 

Emboli  offer  little  of  chemical  interest,  because  of  the  purely  me- 
chanical nature  of  their  origin  and  of  the  effects  they  produce. ^^  An 
exception  exists  in  the  case  of  fat  embolism,  for  the  manner  in  which 
the  fat  is  removed  from  the  blood  has  invited  considerable  specula- 
tion.^^ Part  of  the  fat  is  ehminated  in  the  urine, ^^  but  the  problem 
of  how  it  escapes  from  the  glomerular  capillaries  is  not  satisfactorily 
explained;  large  emboh  undoubtedly  lead  to  rupture  of  the  capillary 

9  Jour.  Med.  Research,  1903  (9),  146. 
1°  Jour.  Med.  Research,  1904  (12),  329;  ibid.,  1906  (14),  541. 

11  Pearce  and  Winne,  Amer.  Jour.  Med.  Sci.,  1904  (128),  669. 

12  Barker,  Jour.  Exp.  Med.,  1908  (19),  343. 

13  Jour.  Exper.  Med.,  1905  (7),  316. 

"Fee  Fleisher  and  Loeb,  Jour.  Biol.  Chem.,  1915  (21),  477. 

1*  Fat  embolism  may  follow  poisoning  with  potassium  chlorate  (Winogradow, 
Virchow's  Arch.,  1907  (190),  92). 

i«  Full  discussion  by  Beneke,  Ziegler's  Beitr.,  1897  (22),  343. 

1'  Discussed  by  Sakaguchi,  (Biochem.  Zeit.,  1913  (48),  1)  who  finds  a  little 
fat  in  the  normal  urine. 


326  DISTURBANCES  OF  CIRCULATION 

walls,  and  probably  some  fat  also  escapes  through  stomata  or  similar 
intercellular  openings.  Fat  may  also  escape  in  the  bile,  and  some  is 
probably  taken  up  by  the  tissue  and  endothelial  cells  by  phagocytosis. 
Beneke  found  that  the  fat  becomes  partly  emulsified  by  the  mechanical 
action  of  the  blood  current,  aided  to  a  slight  extent  by  saponification. 
The  larger  droplets  after  lodging -in  the  capillaries  are  surrounded 
by  leucocytes,  to  which  Beneke  ascribes  an  active  part  in  the  removal 
of  the  fat  as  fine  droplets  by  phagocytic  action.  We  may  well  believe, 
however,  that  the  lipase  of  the  plasma  is  an  important  agent  in  disin- 
tegrating the  emboli,  although  its  action  is  limited  because  of  the  rel- 
atively small  surface  which  the  large  drops  offer  for  attack.  After 
fat  droplets  have  been  taken  into  the  cells,  they  presumably  are  util- 
ized in  metabolism  like  normally  acquired  fat,  as  described  previously. 

The  amount  of  fat  free  in  the  blood  in  fat  embolism  may  be  sur- 
prisingly large.  BisselP*  found  from  2  to  6.5  per  cent,  in  the  venous 
blood  of  several  typical  cases,  although  sometimes  figures  within 
normal  limits  (0.2  to  0.6  per  cent.)  were  found.  The  higher  quantities 
represent  such  a  great  amount  of  free  fat  in  the  blood,  even  without 
considering  the  quantity  held  in  the  capillaries,  that  it  is  scarcely 
possible  for  it  all  to  have  come  from  the  fractured  bones. 

Air  embolism  presents  some  features  of  interest  from  the  chemi- 
cal standpoint,  especially  in  those  cases  following  sudden  decrease  in  at- 
mospheric pressure  in  persons  who  have  been  exposed  for  some  time  to 
pressures  considerably  higher  than  that  of  the  'atmosphere  (diver's 
palsy,  caisson  disease,  etc.).  This  form  of  air  embolism  is  due  to  the 
fact  that  fluids  can  dissolve  much  more  gas  at  high  pressures  than  at 
low  pressures;  consequently  when  the  abnormall}^  great  pressure  to 
which  divers,  caisson  workers,  etc.,  are  subjected  is  too  suddenly  re- 
duced to  that  of  the  atmosphere,  the  excessive  gas  that  was  absorbed 
during  the  period  of  high  pressure  by  the  blood  and  tissue  fluids  is 
released,  and  forms  bubbles  in  the  tissues  and  blood.  The  bubbles  in 
the  nervous  tissues  may  cause  paralyses  of  various  sorts,  or  death; 
those  in  the  blood  may,  if  in  suffi.cient  amount,  cause  serious  or  fatal 
capillary  obstruction.  The  bubbles  consist  chiefly  of  nitrogen,  bo- 
cause  the  power  of  the  blood  to  hold  oxygen  in  combination  is  so  great 
that  not  much  of  this  gas  becomes  freed. ^^  The  body  fluids  of  normal 
persons  contain  about  675  c.c.  of  nitrogen,  all  told,  but  at  22  pounds 
pressure  this  is  increased  to  1350  c.c,  while  but  about  50  c.c.  of  free 
oxygen  would  be  present  (Langlois).  Carbon  dioxitlc  is  so  readily 
combined  in  the  blood  that  none  is  free  even  at  high  pressure,  al- 
though McWhorter-"  reports  that  the  gas  collected  from  t  he  right  side 
of  the  heart  in  a  fatal  case  contained  20  per  cent.  COo  antl  80  per  cent. 

18  Jour.  Aincr.  Med.  Assoc,  lOKi  (G7),  1926. 

"•  Tliis  subject  is  fully  discusscni  by  Leonard  Hill  in  "Recent  Advances  in 
Physiolof^y  and  Biocheniistry;"  London,  lOOti. 

2«  Anicr.  .lour.  Mod.  Sfi.,"l<)10  (LJ'.)),  'M'.i;  Krdinan,  //>((/.,  lOL}  (lb")),  fyiO. 


INFARCTION  327 

nitrogen.  Possibly  some  oxygen  may  also  be  released  from  solution 
during  decompression.-^  At  body  temperature  fats  can  dissolve  five 
times  as  much  nitrogen  as  serum  or  plasma,'^  which  probably  accounts 
for  the  severity  of  the  changes  in  the  nervous  system  with  its  rich 
lipoid  content  and  delicate  structure.  Air  embolism  following  obstet- 
rical operations  or  surgical  operations  about  the  neck  and  chest  pre- 
sents chiefly  mechanical  features, ^^  and  large  quantities  of  air  must  be 
present  to  cause  dangerous  obstruction  to  circulation. ^^  Gas-bubbles 
may  be  produced  in  the  blood  soon  after  death  by  B.  aerogenes  cap- 
sulatus,  but  it  is  not  probable  that  they  are  formed  before  death 
and  cause  air  emboHsm. 

Infarction  . 

The  changes  that  occur  in  infarcted  areas  are  of  much  interest  in 
connection  with  the  study  of  autolysis,  for  the  absorption  of  the  ne- 
crotic tissue  of  aseptic  infarcts  is  purely  a  matter  of  autolj'sis.  Ja- 
coby-^  found  by  ligating  off  a  portion  of  a  dog's  liver,  and  keeping  the 
dog  alive  for  some  time  afterward,  that  in  the  infarcted  tissues  so 
produced  leucine  and  tyrosine  could  be  detected,  just  as  they  are 
found  in  liver  tissue  undergoing  autolysis  outside  of  the  body.  So, 
too,  proteoses  may  appear  in  the  urine  when  any  considerable  amount 
of  tissue  is  cut  off  from  its  blood-supply.  The  processes  of  autolysis 
which  occur  in  ordinary  sterile  infarcts  are,  however,  extremely  slow, 
and  it  is  doubtful  if  enough  of  the  products  are  ever  in  the  blood  or 
urine  at  any  one  time  to  be  detected  or  to  cause  noticeable  effects. 
For  example,  in  an  infarct  of  the  kidney  which  was  known  to  be  al- 
most exactly  fourteen  weeks  old,  there  still  remained  a  layer  of  ne- 
crotic cortex  one  millimeter  thick,  quite  unabsorbed  during  this  time. 
If  we  examine  such  aseptic  infarcts  in  various  stages,  we  get  the  im- 
pression that  the  digestion  is  accomplished  by  leucocytes  acting  on  the 
peripher}^  of  the  infarct,  and  not  entering  the  dead  area  deeply,  pre- 
sumably because  of  a  lack  of  chemotactic  substances  in  the  dead  cells. 
On  the  other  hand,  it  seems  probable  that  the  tissue  enzymes  them- 
selves play  an  important  part  in  the  autolysis,  for  if  we  implant  into 
animals  pieces  of  tissue  in  which  the  enzymes  have  been  destroyed  by 
heating  to  boiling,  it  will  be  found  that  the  cells  and  their  nuclei  re- 
main unaffected  for  many  weeks;  whereas  if  sterile  unheated  pieces 
of  tissue  in  which  the  enzymes  are  still  active  are  implanted,  they  lose 
their  nuclear  stain  and  begin  to  disintegrate  relatively  soon,  without 
apparent  participation  by  the  leucocytes.-^     Ribbert-^  found  that  in 

21  HUl  and  Greenwood,  Proc.  Royal  Soc.  (B),  1907  (79),  284. 

"Vernon,  ibid.,  p.  366;  Quincke,  Arch.  exp.  Path.  u.  Pharm.,  1910  (62),  -164. 

-^  Review  of  literature  by  Wolff,  Virchow's  Archiv.,  1903  (174),  454. 

-^  See  Hare,  Anier.  Jour.  Med.  Sciences,  1902  (124),  843. 

"  Zeit.  phvsiol.  Chem.,  1900  (30),  149. 

2«  Wells,  Jour.  Med.  Research,  1906  (15),  149. 

"  Virchow's  Arch.,  1899  (155),  201. 


328  DISTURBANCES  OF  CIRCULATION 

experimentally  produced  anemic  infarcts  in  the  kidneys  of  rabbits  the 
nuclei  retain  their  staining  property  well  for  nearly  twenty-four  hours, 
becoming  then  small  and  deeply  stained,  undergoing  karj^orrhexis, 
and  in  large  part  disappearing  from  the  convoluted  tubules  inside  of 
forty-eight  hours,  although  some  nuclei  may  persist  in  the  glomerules 
for  three  or  more  days.  In  human  infarcts,  Ribbert  believes,  the 
process  goes  on  faster,  for  he  has  osberved  here  a  loss  of  nuclei  within 
twenty-four  hours.  These  nuclear  changes  undoubtedly  depend  upon 
autolysis,  but  it  is  probable  that  the  enzymes  concerned  reside  in  the 
cytoplasm  rather  than  in  the  nucleus,  for  I  have  observed  that  cells 
of  lymphoid  type,  with  practically  no  cytoplasm,  generally  retain 
their  nuclear  stain  much  longer  than  cells  with  more  cytoplasm;  this 
is  particularly  noticeable  in  splenic  infarcts,  where  the  ]\Ialpighian 
corpuscles  retain  their  staining  affinities  much  longer  than  the  pulp 
elements.  Whether  the  destruction  of  the  nuclei  is  accomplished 
by  the  ordinary  intracellular  proteases,  or  by  special  nucleoprotein- 
splitting  enzymes  (nuclease, ^^  etc.),  remains  to  be  determined.  It  is 
quite  possible,  however,  that  the  first  changes  consist  of  a  splitting 
of  the  nucleoproteins  of  the  nucleus  by  the  autolytic  enzymes,  liber- 
ating the  nucleic  acid,  which  gives  the  nuclei  the  characteristic  intense 
staining  with  basic  dyes  (pycnosis)  observed  in  areas  of  early  anemic 
necrosis.  The  nucleic  acid  may  then  be  further  decomposed  by  the 
nuclease  or  similar  enzymes.  Taken  all  together,  then,  it  would  seem 
that  the  nuclear  and  cellular  alterations  that  make  up  the  character- 
istic picture  of  anemic  necrosis  are  brought  about  by  the  intracellular 
enzymes — 'an  autolytic  process.  The  removal  of  the  dead  substance, 
however,  seems  to  be  accomplished  rather  by  the  invading  leucocytes, 
through  heterolysis.  The  relatively  small  part  taken  by  the  intracel- 
lular enzymes  may  possibly  be  due  to  the  seeping  through  them  of  alka- 
line blood-plasma,  for  autolytic  enzymes  are  not  active  in  an  alkaline 
medium;  the  leucocytic  enzymes,  however,  act  best  in  an  alkaline 
medium. 2^ 

About  the  periphery  of  infarcts  is  usually  observed  more  or  less  fat 
deposition  (Fischler),^"  particularly  in  the  endothelial  cells  (Ribbert). 
This  is  not  peculiar  to  infarcts,  however,  for  Sata^'  found  a  similar 
peripheral  fatty  metamorphosis  common  to  all  necrotic  areas.  The 
basis  of  this  is  possibly  the  persistence  of  the  cell  lipase,  wliich  syn- 
thesizes fatty  acid  and  glycerol  diffusing  into  the  necrotic  area  with 
the  plasma,  unchecked  by  normal  oxidative  destruction  of  these 
substances.     (See  "Fatty  Degeneration,"  Chap,  xvi.) 

Hemorrhagic  infarcts  offer  in  addition  to  the  changes  conunon 
to  anemic  infarcts,  the  alterations  occurring  in'  the  blood-corpuscles. 

"»  Sachs,  Zoit   physiol.  Chcm.,  1905' (4C), '337;  Schittonhclni,  ibid.,  354.' 
^^  More  fully  discussed  by  Wells,-"  loc.  cit.,  and  under  necrosis,  Chap.  xv. 
30  Cent.  f.  Path.,  1«)()'2  (13),  417. 
"  Ziegler's  lieitr.,  1900  (28),  461. 


INFARCTION  329 

Panski''^  found  that  after  ligation  of  the  splenic  vein  of  dogs  the  red 
corpuscles  begin  to  give  up  their  hemoglobin  in  about  three  hours. 
After  twelve  hours  fibrin  formation  begins  in  the  tissues,  the  corpus- 
cles continue  to  give  up  hemoglobin  and  become  cloudy  in  appearance. 
Later,  iron-containing  pigment  is  formed  in  the  cells  beneath  the  cap- 
sule, but  in  the  deeper  tissue  even  the  iron  normally  present  in  the 
spleen  tissue  seems  to  disappear ;^^  this  possibly  depends  upon  the 
fact  that  pigment  reacting  for  iron,  hemosiderin,  is  formed  only  in 
living  cells  under  the  influence  of  oxygen,  or  it  may  be  that  acids 
formed  during  autolysis  dissolve  it.  During  autolysis  in  vitro,  how- 
ever, Corper^*  found  no  evidence  of  removal  of  iron  from  insoluble 
or  coagulable  compounds.  The  hemolysis  is  probably  produced  either 
by  the  action  of  autolytic  products,  which  are  notoriously  hemolytic, 
or  perhaps  also  by  direct  attack  of  tissue  and  blood  proteases  upon 
the  corpuscles. 

Other  retrogressive  changes  that  may  occur  in  infarcts,  such  as  sep- 
tic softening  and  calcification,  are  not  greatly  different  from  the  same 
processes  occurring  in  other  conditions,  and  will  be  considered  with 
the  discussion  of  these  processes. 

'2  "Untersuchungen  iiber  den  Pigmentgehalt  der  Stauungsmilz,"  Dorpat,  1890. 
"  See  also  M.  B.  Schmidt,  Cent.  f.  Path.,  1908  (19),  416. 
="•  Jour.  Exper.  Med.,  1912  (15),  429. 


CHAPTER  XIV 
EDEMA' 

As  the  term  edema  indicates  the  excessive- accumulation  of  lymph 
(which  may  be  either  normal  or  modified  in  composition)  in  the  cells, 
intercellular  spaces,  or  serous  cavities  of  the  body,  the  problems  of 
edema  are  inseparably  connected  with  the  consideration  of  the  proc- 
esses of  physiological  formation  and  removal  of  lymph.  For  many 
years  the  study  of  these  processes  has  been  a  favorite  field  of  investi- 
gation by  physiologists,  and  the  great  battle-place  of  the  "vitalistic" 
and  ''mechanistic"  schools;  and  to  this  day  the  forces  that  determine 
the  formation  of  lymph  and  its  subsequent  absorption  have  not  been 
completely  understood.  By  the  application  of  the  principles  of  phys- 
ical chemistry  to  the  problem,  however,  great  advances  have  recently 
been  made,  which  seem  to  render  our  understanding  of  both  Ijanph- 
formation  and  its  pathological  accumulation  in  the  tissues  nuicli 
clearer  and  more  nearly  accurate  than  they  were  before.  We  shall 
first  consider,  therefore  the  physiological  formation  of  lymph,  before 
taking  up  the  subject  of  edema. 

Composition  of  Lymph. — Lymph  consists  of  material  derived  from  two  chief 

sources.  The  greater  part  consists  of  fluid  passing  out  of  the  capillaries  into 
the  tissue  spaces;  here  it  is  modified  by  the  addition  of  products  of  nietabolism 
derived  from  the  tissue-cells,  and  by  the  subtraction  of  materials  that  the  cells 
utilize  in  their  metabolism.  It  is,  therefore,  essentially  a  modified  blood  plasma, 
and  the  modifications  the  plasma  undergoes  are  so  slight,  that,  under  ordinary 
conditions,  lymph  shows  on  analysis  no  considerable  differences  from  blood 
plasma,  except  a  relative  poverty  in  proteins,  due  chifly  to  the  impermeability 
of  the  capillary  walls  for  colloids.  Its  quantitative  composition  varies  greatly^ 
depending  upon  the  conditions  under  which  it  is  collected,  whether  during  activitja 
or  rest,  etc.     The  following  tables  of  analyses  have  been  collected  by  Hammarsten: 

12                     3  4 

Water 939.9           934.8           957.6  955.4 

Solids 60.1             65.2             42.4  44.6 

Fibrin 0.5               0.6               0.4  2.2 

Albumin 42.7  42.8  34.7  1 

Fat,  Cholesterol,  Lecithin 3.8               9.2            [  35.0 

Extractive  bodies 5.7  4.4  I 

Salts 7.3               S.2               7.2  7.5 

1  and  2  are  analyses  of  lymph  from  the  thigh  of  a  woman,  3  is  from  the  contents 

of  sac-like  dilated  vessels  of  the  spermatic  cord,  4  is  lymph  from  the  neck  of  a  colt. 

'  A  complete  bibliography  is  given  by  Meltzor,  .\merican  Medicine,  1001  (S), 
19  cl  sctj.;  also  by  Klemensiewicz,  in  Krelil  ami  Marchaml's  ll;iiull)uch  d.  allg. 
Path.,  1912,  II  (i),  311;  Magnus,  llandbuch  d.  Hiochem.,  1908,  11  {2),  99;  C.er- 
hartz,  ihifl.,  j).  116. 

330 


FORMATION  OF  LYMPH  331 

Chyle  differs  from  lymph  chiefly  in  the  presence  of  large  quantities  of  fat; 
during  starvation  the  lymph  and  the  chyle  are  of  practically  the  same  composition. 

Normal  lymph  contains  much  less  fibrinogen  than  does  the  blood  plasma,  and 
hence  coagulates  slowly.  Lipase  and  other  enzymes  have  been  found  in  the  lymph, 
as  in  the  plasma.  The  products  of  tissue  metabolism  added  to  the  lymph  by 
the  cells  may  render  it  toxic  (Asher  and  Barbera').  Under  pathological  condi- 
tions the  lymph  may  be  greatly  altered,  becoming  poorer  in  solids  under  some 
conditions  of  edema,  and  becoming  rich  in  proteins  and  blood-corpuscles  under 
inflammatory  conditions,  until  it  partakes  of  the  characteristics  of  an  inflam- 
matory exudate  (see  analyses  of  transudates  and  exudates). 

An  important  fact  to  consider  is,  that  of  the  entire  water  of  the 
body  but  about  one-tenth  is  in  the  blood.  About  two-thirds  of  the 
entire  weight  of  the  body  is  water,  which  is  mostly  in  the  cells  and 
tissues,  firmly  bound  by  the  colloids,  only  an  unknown  but  smaller 
portion  being  as  free  movable  fluid,  and  even  here  always  associated 
with  more  or  less  colloid.  A  body  weighing  60  kilos  will,  therefore, 
have  40  kilos  of  water,  of  which  but  about  4  kilos  is  blood. 

FORMATION  OF  LYMPH^ 

Filtration  Theory. — The  simplest  possible  conception  of  lymph 
formation  is  that  it  is  merely  the  result  of  filtration  of  the  liquid  con- 
stituents of  the  blood  through  the  capillary  walls  under  the  influence 
of  the  blood  pressure.  This  "filtration  theory"  was  supported  origi- 
nally by  Ludwig,  and  it  was  a  prominent  factor  in  the  early  appli- 
cations of  mechanical  principles  to  biological  processes.  In  support 
of  this  theory  were  advanced  the  results  of  numerous  experiments  in 
which  it  was  shown  that  increasing  the  blood  pressure  by  means  of 
ligating  the  veins,  or  by  causing  arterial  dilata  ion,  resulted  in  an  in- 
■  crease  of  the  lymph  flowing  out  of  the  lymph-vessels  of  the  part. 
Also,  when  the  blood  pressure  is  raised  by  epinephrine  or  by  other 
means,  a  large  proportion  of  the  fluid  leaves  the  blood  vessels;  con- 
versely, when  the  blood  pressure  is  suddenly  lowered  by  hemorrhage 
there  is  a  rapid  passage  of  fluid  from  the  tissues  into  the  blood.  The 
experimental  results  were  not  always  favorable  to  the  theory,  how- 
ever, particularly  in  the  experiments  in  which  blood  pressure  was 
raised  by  arterial  dilatation;  often  the  flow  of  lymph  was  Kttle  in- 
creased, even  when  the  arterial  flow  and  pressure  were  greatly  in- 
creased. iSlevertheless,  the  filtration  theory  held  for  many  years,  not 
only  as  an  explanation  of  lymph  formation,  but  also  as  an  explanation 
of  urinary  secretion  and  of  the  secretion  by  other  organs.  It  was 
only  within  a  comparatively  short  time  that  it  became  clear  that  filtra- 
tion alone  could  not  account  for  all  the  phenomena  of  secretion.  For 
example,  in  many  lower  forms  with  undeveloped  circulatory  systems, 
and  almost  no  blood  pressure,  secretion  goes  on  vigorously;  the  pres- 
sure of  glandular  secretions  may  be  much  higher  than  the  blood 
pressure  within  the  capillaries;  the  activity  of  secretion  is  by  no  means 

2  Zeit.  f.  Biol.,  1898  (36),  154. 

^  See  review  by  Asher,  Biochem.  Centralblatt,  1905  (4),  1. 


332  EDEMA 

in  proportion  to  blood  pressure,  etc.  If  in  glandular  secretion,  there- 
fore, fluids  are  removed  from  the  blood  and  transferred  into  an  ex- 
cretory duct  through  the  action  of  some  force  other  than  that  of  the 
blood  pressure,  it  is  probable  that  lymph  formation  is  equally  inde- 
pendent of  blood  pressure.  On  this  basis  Heidenhain  advanced 
his — 

Secretory  theory  of  lymph  formation,  in  which  he  suggested  that 
lymph  is  the  product  of  an  active  secretion  by  the  endothelial  cells 
of  the  capillaries,  just  as  saliva  is  the  product  of  the  activity  of  the 
glandular  cells.  He  showed  that  certain  chemical  substances  may 
stimulate  lymph  flow,  independent  of  blood  pressure,  just  as  pilocar- 
pine and  other  drugs  may  stimulate  the  secretion  of  saliva.  These 
lymph-stimulating  substances,  which  he  named  lymphagogues,  fall  into 
two  distinct  classes.  One  which  includes  such  substances  as  peptone, 
leech  extract,  strawberry  juice,  extracts  of  crayfish,  mussel  or  oysters, 
and  numerous  other  tissue  extracts,  are  characterized  by  causing  the 
secretion  of  a  lymph  which  is  rich  in  proteins,  even  richer  in  proteins 
than  the  blood  plasma;  and,  furthermore,  there  is  no  simultaneous 
increase  in  urinary  secretion.  Heidenhain  considered  that  these  sub- 
stances caused  lymph  secretion  by  stimulating  the  capillary  endothe- 
hum  in  a  specific  manner;  as  they  caused  no  appreciable  rise  in  blood 
pressure  the  increased  lymph  secretion  certainly  could  not  be  attrib- 
uted to  filtration.  This  independence  of  the  lymph  flow  of  blood 
pressure  is  most  conclusively  shown  by  posttriortem  lymph  secretion; 
for  example,  Mendel  and  Hooker'*  observed  lymph  flow  for  four  hours 
after  death,  in  a  dog  that  had  received  an  injection  of  peptone  eight 
minutes  before  being  killed.^ 

The  second  class  of  lymphagogues  includes  crystalloidal  substances, 
such  as  sugar,  urea,  and  salts. ^  Lymph  secreted  under  the  influence 
of  these  substances  is  poorer  in  protein  than  ordinary  h'mph,  and  at 
the  same  time  an  increased  urinary  secretion  is  produced.  With 
these  crystalloidal  lymphagogues  the  amount  of  effect  is  in  inverse 
proportion  to  their  molecular  weight,  which  means  that  their  effects 
depend  upon  the  number  of  molecules  in  solution  rather  than  upon 
their  nature;  in  other  words,  the  stimulation  of  Ij^mph  by  crystalloids 
is  dependent  upon  the  osmotic  pressure  of  the  crj'stalloids.  Heiden- 
hain explained  their  action  as  follows:  The  crystalloids  are  secreted 
into  the  lymph-spaces  by  the  action  of  the  capillary  endothelium,  and 
there,  owing  to  their  raising  osmotic  pressure,  cause  a  flowing  of 
water  out  of  the  vessels.  The  difficulty  here  is  to  explain  why  the 
crystalloids  while  still  in  the  vessels  do  not  attract  the  fluids  from 

*  Amer.  Jour,  of  Physiol.,  1902  (7),  380. 

'  A  fact  not  sufficiently  taken  into  account  is  that  blisters  filled  with  serum, 
i.  e.,  an  inflaniinatory  edema,  may  be  produced  in  dead  bodies  bv  liurns  or  scalds. 
(See  Leers  and  Raysky,  Virchow's,  Arcli.,  1909  (197),  324). 

*  The  action  of  many  other  substances  has  been  investigated  bv  Vanagawa, 
Jour.  Pharmacol.,  191()  (9),  75. 


FORMATION  OF  LYMI'II  333 

the  lymph-spaces  into  the  blood,  and  so  cause  rather  a  lessened  lymph 
secretion. 

While  admitting  that  in  pathological  conditions  {e.  g.,  passive  con- 
gestion) pressure  and  filtration  7najj  play  an  important  part,  Heiden- 
hain  considered  that  an  active  secretion  by  the  endothelial  colls  is  the 
chief  factor  in  the  normal  formation  of  lymph.  The  means  by  which 
the  cells  perform  this  function  was  unknown;  it  was  considered  as  an 
example  of  "vital  activity,"  Heidenhain  meaning  by  this  term  such 
chemical  and  physical  forces  of  living  cells  as  are  unknown  or  not 
understood  at  the  present  time,  rather  than  any  metaphysical  concep- 
tion of  living  matter,  such  as  many  vitalists  assume. 

Other  observers,  corroborating  Heidenhain's  results  for  the  most 
part,  have  modified,  or  amplified  his  theory.  Asher  and  his  collabo- 
rators, for  example,  ascribe  the  work  done  in  causing  lymph  forma- 
tion to  the  cells  of  the  various  'issues  and  organs,  rather  than  to  those 
of  the  capillar}'-  wall.  The  increased  flow  of  lymph  from  the  salivary 
gland  that  occurs  during  its  activity  they  consider  due  to  the  work 
of  the  gland  cells,  and  its  function  the  removal  of  products  of  metab- 
olism. The  action  of  such  a  lymphagogue  as  peptone  they  ascribe  to 
its  stimulation  of  cellular  activity,  particularly  in  the  liver,  where  it 
causes  an  increased  formation  of  bile.  Gies^  and  Asher  also  ob- 
served that  after  an  injection  of  crystalloidal  lymphagogues,  such  as 
sugar,  a  prolonged  flow  of  lymph  occurred  after  the  death  of  the 
animal,  proving  completely  that  such  lymphagogic  action  is  inde- 
pendent of  blood  pressure. 

Potocytosis. — In  explanation  of  the  process  by  which  the  cells,  whether  en- 
dothelial or  tissue-cells,  pass  fluids  through  themselves  from  one  place  to  another, 
Meltzer^  has  made  an  interesting  suggestion,  as  follows:  Considering  the  prop- 
erty of  endothelial  cells  to  act  as  phagocytes,  MacCallum*  has  shown  that  solid 
granules  (e.  g.,  coal  pigment,  carmin)  are  taken  through  the  walls  of  the  lymphat- 
ics by  the  phagocytic  activity  of  their  endothelial  cells.  Meltzer  suggests  that  in 
a  similar  way  the  endothelial  cells  may  transport  through  the  vessel-walls  not 
only  solid  particles,  but  also,  by  the  same  mechanism,  substances  in  solution; 
and  for  this  hypothetical  process  he  suggests  the  name  "potocytosis."  There  can 
be  little  question  that  cells  do  take  up  substances  in  solution,  and  sometimes  this 
is  done  in  an  apparently  selective  manner;  e.  g.,  the  taking  up  of  bacterial  toxins 
and  vegetable  poisons  in  the  peritoneal  cavity  by  the  leucocytes.  Presumably 
the  mechanism  of  "potocytosis"  is  not  different  from  that  of  phagocytosis,  chemo- 
tactic  forces  determining  the  occurrence  of  the  process.  No  experimental  evi- 
dence has  been  advanced  as  yet  for  this  very  plausible  hypothesis. 

Permeability  of  Capillaries. — In  explanation  of  the  variabihty 
in  the  amount  and  composition  of  the  lymph.  Starling^  has  introduced 
the  factor  of  altered  permeability  of  the  capillary  walls,  which  pre- 
sumably depends  upon  the  number  and  size  of  the  pores.  He  found 
that  normally  the  lymph  coming  from  the  lower  extremities  contains 

7  Amer.  Jour.  Physiol.,  1900  (3),  p.  xix;  Zeit.  f.  Biol.,  1900  (40),  207. 

«  Johns  Hopkins  Hosp.  Bull.,  1903  (14),  1. 

'Lancet,  1896  (i).  May  9,  et  seq.;  Schafer's  Text-book  of  Physiology,  vol.  1. 


334  EDEMA 

only  2  per  cent,  to  3  per  cent,  of  proteins,  while  lymph  from  the  intes- 
tines contains  4  per  cent,  to  6  per  cent.,  and  lymph  from  the  liver  con- 
tains 6  per  cent,  to  8  per  cent,  of  proteins;  hence  he  considers  that  the 
liver  capillaries  are  the  most  permeable,  i.  e.,  have  the  largest  pores, 
so  that  more  of  the  large  colloid  molecules  can  escape  from  them.  The 
effect  of  lymphagogues  of  the  first  class  (peptones,  etc.)  he  attributes 
to  their  poisonous  properties,  and  the  consequent  injury  to,  and  altera- 
tions in,  the  capillary  wall.  The  crystalloidal  lymphagogues,  he 
believes,  act  by  first  attracting  fluids  from  the  tissues  into  the  blood 
with  a  resulting  "hydremic  plethora,"  which  in  turn  leads  to  in- 
creased blood  pressure  and  consequent  filtration  of  a  watery  fluid 
out  of  the  vessels.  He  considers,  therefore,  that  the  amount  and 
quality  of  the  lymph  produced  in  any  part  are  determined  solely  by 
two  factors,  the  intracapillary  blood  pressure  and  the  permeability  of 
the  capillary  walls. 

In  connection  with  this  question  of  the  permeability  of  the  capil- 
lary walls,  Meltzer  suggests  that  the  contractility  and  irritability  of 
the  endothelium  may  be  a  potent  factor  in  determining  the  size  of  the 
pores  in  the  capillary  walls.  When  in  a  tonic  condition,  the  endothe- 
lium is  firmly  contracted  about  the  pores,  keeping  their  size  small; 
when  the  endothelial  cells  become  relaxed  by  any  cause,  such  as  poi- 
sons, high  blood  pressure,  poor  nourishment,  etc.,  the  pores  are  en- 
larged, and  increased  escape  of  fluid  results.^"  It  must  be  borne  in 
mind,  however,  that  most  histologists  do  not  now  admit  that  capillary 
walls  contain  pores. 

M.  H.  Fischer  holds  that  the  endothelial  cells  undergo  changes  in 
consistency  through  changes  in  the  affinity  of  the  cell  colloids  for  wa- 
ter; especially  under  the  influence  of  acids  the  endothelium  ma}'  be- 
come much  more  fluid  and  of  greater  permeability.  Adolf  Oswald^" 
says  that  the  normal  capillary  wall  is  somewhat  permeable  for  the 
less  viscous  blood  proteins  (albumin  and  pseudoglobulin),  and  in  in- 
flammation this  permeability  becomes  increased  so  that  the  more  vis- 
cous euglobulin  and  fibrinogen  can  pass  through. 

Osmotic  Pressure. — Still  another  possible  factor  in  causing  fluid 
to  leave  the  vessels  is  osmotic  pressure.  Heidenhain  refers  to  this 
cause  the  transudation  produced  by  crystalloid  lymphagogues,  al- 
though in  a  rather  unsatisfactory  manner.  As  a  result  of  the  more 
recent  studies  of  physical  chemistry,  and  its  application  to  biological 
processes,  we  have  learned  to  appreciate  the  importance  of  osmotic 
pressure  in  cell  activities  (see  Introductory  Chapter),  and  in  the 
question  of  lymph  formation  it  occupies  a  particularly  important 
place.  We  may  consider  it  as  follows:  In  the  blood  we  have  certain 
proportions  of  readily  diffusible  crystalloids  and  of  non-diffusible 

""  Evidence   of    the    contractility    of   capillary  walls  is  discussed  by  Krogh, 
Jour.  Physiol,  1919  (52),  457. 

">  Zeil.  f.  exp.  Path.,  1910  (8),  226.  ^ 


FORMATION  OF  LYMPH  335 

colloids.  If  no  metabolic  processes  were  going  on  in  the  tissues,  wo 
should  have  the  diffusible  substances  leaving  the  vessel-walls  (leaving 
out,  for  the  present,  any  question  of  secretory  activity  on  the  part  of 
the  endothelium)  until  an  osmotic  equilibrium  is  established  in  the 
tissues  and  in  the  blood.  As  a  matter  of  fact,  however,  the  blood  pro- 
teins are  not  absolutely  non-diffusible,  but  small  (juantities  do  pass 
through  the  capillary  walls,  and  so  lymph  under  such  a  hypothetical 
condition  would  consist  of  a  mixture  of  the  same  osmotic  concentration 
as  the  blood  plasma,  with  about  the  same  proportion  of  crystalloids, 
but  a  smaller  proportion  of  proteins;  this,  it  will  l)e  noticed,  is  just 
about  the  composition  of  normal  lymph.  During  life,  however,  the 
cells  of  the  tissues  are  causing  metabolic  changes  in  these  lymphatic 
constituents,  and  these  changes  consist  chiefly  in  breaking  down  large 
molecules  of  proteins,  carbohydrates,  and  fats  into  much  smaller 
molecules.  Now  the  osmotic  pressure  of  a  solution  is  dependent  upon 
the  yiumher  of  molecules  and  ions  it  contains,  hence  by  breaking  down 
these  few  large  molecules  with  very  little  osmotic  pressure  into  many 
small  molecules,  the  osmotic  pressure  in  these  cells  and  tissues  be- 
comes raised  above  that  of  the  blood-vessels,  and  consequently  water 
flows  out  of  the  vessels  because  of  the  increased  pressure.  We  see  here 
the  probable  explanation  of  the  stimulating  influence  of  metabolic 
products  upon  the  formation  of  lymph,  noted  by  Hamburger,  Heiden- 
hain,  and  others.  For  suggesting  and  urging  the  importance  of  osmo- 
tic pressure  in  the  formation  of  lymph  we  are  indebted  particularly 
to  Heidenhain,  v.  Koranyi,^^  J.  Loeb,^^  and  Roth.'^  Loeb  shows  very 
clearly  the  relative  greatness  of  the  water-driving  force  of  osmotic 
pressure  as  compared  to  that  of  blood-pressure,  by  his  statement  that 
the  osmotic  pressure  of  a  physiological  salt  solution  is  about  4.9  atmos- 
pheres, which  is  twenty  times  as  great  as  the  blood  pressure  with  which  we 
have  to  do  in  ordinary  physiological  experiments.  In  varying  osmotic 
conditions  we  may  readily  see  an  explanation  for  the  increased  lymph 
flow  that  occurs  during  tissue  activity;  namely,  it  is  due  to  the  in- 
creased formation  of  metabolic  products.  Many  of  the  lymphagogues 
may  act  similarly  by  stimulating  metabolic  activity,  with  resulting  in- 
crease in  the  formation  of  osmotic  pressure-raising  products  of  metab- 
olism in  the  organs;  e.  g.,  the  increased  lymph  flow  from  the  thoracic 
duct  that  follows  stimulation  of  hepatic  activity  by  injection  of  pep- 
tone (Heidenhain)  or  ammonium  tartrate  (Asher  and  Busch).'*  As 
we  shall  see  later  in  considering  edema,  osmotic  pressure  may  play  an 
important  part  in  the  pathological  formation  of  lymph.  It  must  be 
admitted,  however,  that  there  are  many  difficulties  in  the  way  of 
accepting  unqualifiedly  the  original  views  as  to  the  importance  of 

"  Zeit.  f.  klin.  Med.,  1897  (33),  1;  1898  (34),  1.- 

12  Pfliiger's  Arch.,  1898  (71),  457. 

13  Englemann's  Arch.,  1899,  p.  416. 
"  Zeit.  f.  Biol.    1900  (40),  333. 


336  EDEMA 

osmotic  pressure  in  lymph  formation. ^^  For  example,  the  lymph 
contains  more  chlorides  and  may  have  a  much  higher  osmotic 
pressure  than  the  serum  of  the  same  animal  (Hamburger,  Carlson, 
et  al.).'' 

Variable  Capacity  of  Colloids  for  Water. — Colloids  of  the  type  of  the 
tissue  proteins,  i.  e.,  hydrophil  colloids,  imbibe  water  with  great  avid- 
ity, until  a  certain  proportion  of  water  is  present,  the  proportion 
varying  under  different  conditions.  The  importance  of  this  force  in 
the  production  of  edema  and  related  processes  was  first  pointed  out 
by  Martin  H.  Fischer,  and  has  been  developed  extensively  by  him." 
The  amount  of  water  which  a  given  hydrophil  colloid,  such,  for  exam- 
ple, as  gelatin,  or  fibrin,  will  take  up,  is  greatly  modified  by  the  reac- 
tion of  the  solution  and  by  its  content  of  electrolytes.  Very  small 
concentrations  of  acids  or  alkalies  will  greatly  increase  the  amount 
of  water  absorbed,  while  salts  reduce  it,  and  the  different  acids  and 
salts  vary  in  their  effects;  thus  hydrochloric  acid  causes  a  greater 
swelling  of  colloids  than  a  corresponding  strength  of  sulphuric  acid, 
and  calcium  chloride  depresses  the  swelhng  more  than  potassium 
chloride.  The  effect  of  the  salts  is  made  up  of  their  constituent  ions. 
Non-electrolytes  have  relatively  little  effect.  The  forces  developed 
by  this  affinity  of  colloids  for  water  are  enormous;  thus,  to  prevent 
the  taking  up  of  water  by  starch  requires  a  pressure  of  over  2500  at- 
mospheres, dried  gelatin  will  take  up  25  times  its  weight  of  water, 
and  fibrin  as  much  as  forty  times.  Different  colloids  differ  greatly  in 
their  affinity  for  water  and  in  the  way  in  which  this  afl&nity  is  modi- 
fied by  electrolytes,  and  change  in  a  colloid  may  greatly  alter  its 
capacity  for  swelling;  thus,  jS-gelatin,  which  can  be  formed  from  ordi- 
nary gelatin  by  the  action  of  proteolytic  enzymes,  has  greater  capacity 
for  swelling  than  the  original  gelatin.  Gies  especially  lays  stress  on 
this  factor,  that  is,  the  alterations  of  the  hydrophilic  tendencies  of  the 
tissue  colloids  by  enzymes.'^  In  the  plant  world  we  find  striking 
examples  of  this  character;  thus,  the  succulence  of  some  plants  results 

15  Gunzberg  (Arch.  ncerlandphysioL,  1918  (2),  364)  states  that  the  passage  of 
water  into  the  intercellular  spaces  is  due  to  the  electrical  properties  of  the  mem- 
brane separating  the  circulating  fluid  from  the  tissues.  The  element  potassium 
and  the  ioas  H  and  OH  play  an  important  part  in  this  electrical  osmosis  which  is 
able  to  drive  the  fluid  in  the  opposite  direction  to  osmotic  pressure.  Thus,  a 
dialyzing  sack  containing  Ringer  solution  minus  K  immersed  in  Ringer  solution 
loses  weight.  Perfusion  of  frogs  with  Ringer  solution  minus  K  produces  marked 
edema. 

16  Amer.  Jour.  Physiol.,  1907  (19),  3G0;  1908  (22),  91. 

1' See  Fischer's  Monograph,  "Oedema  and  Nephritis,"  New  York,  1915;  also 
numerous  articles  in  the  Zeit.  f.  Chem.  u.  Ind.  d.  Kolloide.  An  especially  thor- 
ough discussion  of  this  theory  is  contained  in  the  Biochemical  Bulletin,  Vol.  I., 
giving  a  bibliography  of  Fischer's  work,  together  with  articles  on  Gies'  observa- 
tions on  the  modification  of  the  hydrophilic  tendency  of  proteins  by  enzyme 
action. 

1*  A  definite  and  clear-cut  example  of  the  swelling  of  a  tissue  under  the  in- 
fluence of  acid  of  metabolic  origin  is  shown  in  the  muscle  cell  in  Zenker's  waxy 
degeneration  (Wells,  Jour.  Exper.  Med.,  1909  (11),  1). 


FORMATION  OF  DM  I'll  337 

from  the  conversion  of  i)ol3'-.sa('('li;iri(l(',s  witli  little  li^'di'alion  capacity 
into  hyclrophilic  pentosans  and  mucilages.'^ 

On  the  basis  of  the  facts  briefly  suniniarized  above,  the  proportion 
of  water  present  in  any  cell  or  in  anj^  fluid  of  the  body  which  contains 
colloids,  is  assumed  to  be  determined  by  certain  factors,  namely  (1) 
the  character  of  the  colloids  themselves;  (2)  the  proportion  and  na- 
ture of  acids  or  alkalies  present  in  the  fluids  in  and  about  the  colloids; 
(3)  the  i^-oportion  and  nature  of  the  salts.  All  these  factors  are 
changeable,  and  therefore  the  amount  of  water  present  in  the  cell  or 
fluid  varies  accordingly.  Thus,  if  a  cell  through  its  metabolism  de- 
velops from  such  a  non-electrolyte  as  sugar  (which  has  no  consider- 
able effect  on  the  water  content  of  the  protoplasm),  an  organic  acid, 
such  as  lactic  acid,  which  has  a  large  effect  in  increasing  the  affinity 
of  the  colloids  for  water,  the  cell  will,  presumably,  take  on  more  water, 
perhaps  to  a  degree  to  cause  intracellular  edema.  The  acids  diffusing 
from  the  cell  into  the  intercellular  spaces  or  into  the  lymph  will 
cause  equally  well  an  increased  affinity  for  water  in  the  colloids  here 
present,  leading  to  intercellular  edema.  Conversely,  neutralization 
of  acids  present  in  a  colloidal  solution,  by  alkaline  salts  brought  by 
the  blood,  will  decrease  the  affinity  of  the  colloids  for  water  which 
will  escape  from  the  colloids  as  they  shrink.    • 

This  theory,  which  introduces  a  hitherto  unappreciated  factor  into 
the  considerations  of  lymph  formation  and  edema,  is  of  the  utmost 
importance.  It  practically  eliminates  osmotic  pressure,  also  the  cell 
membranes  so  essential  for  the  efficiency  of  this  force,  and  in  view  of 
the  difficulties  that  have  arisen  in  trying  to  fit  the  cell  membrane 
hypothesis  and  osmotic  pressure  to  many  facts  of  normal  and  patho- 
logical biology,  an  alternative  hypothesis  is  welcome.  As  pointed  out 
above,  the  forces  involved  in  the  swelling  of  colloids  are  so  large  as  to 
be  of  great  significance,  and  the  amounts  of  electrolytes  necessary 
to  cause  considerable  variations  in  colloidal  swelling  are  not  more  than 
can  be  present  under  normal  and  pathological  conditions;  conse- 
quently the  possible  influence  of  colloidal  swelhng  must  be  taken 
into  account  in  all  consideration  of  pathological  processes.  Whether 
or  not  it  is  capable  of  as  universal  application  as  Fischer  mamtains, 
remains  to  be  demonstrated,  and  there  are,'  indeed,  some  facts  that 
do  not  seem  to  be  in  harmony  with  this  theory. 

Summary. — We  see  from  the  above  discussion  that  numerous  the- 
ories have  been  advanced  to  explain  the  normal  formation  of  lymph, 
and  as  their  basis  exist  several  different  possible  factors.  Filtration, 
active  secretion  by  the  capillary  endothelium,  attraction  by  the  tissue- 
cells,  osmosis  in  response  to  formation  of  crystalloids  outside  the  ves- 
sels, and  changes  in  the  affi.nity  of  colloids  for  water;  all  have  been 
shown  to  be  possible  causes  of  lymph  formation.  It  is  highly  probable 
that  in  a  certain  wa}'  all  are  involved,  particularly  if  we  accept  the 

19  MacDougal  and  Spoehr,  Plant  World,  1918  (21),  245. 

22 


338  EDEMA 

view  of  the  physical  school  that  "secretion"  and  "attraction"  by 
the  cells  are  merely  the  outcome  of  physical  forces;  the  causes  of 
lymph  formation  then  reduce  themselves  to  absorption,  filtration  and 
diffusion.  There  has  been,  until  recently,  no  question  but  that  lymph 
does  escape  from  the  vessels  through  simple  filtration,  for  the  pressure 
inside  the  capillaries  is  presumably  greater  than  outside,  the  capillary 
walls  are  not  water-tight  and  they  are  not  impermeable  to  the  sub- 
stances dissolved  in  the  plasma.^"  Likewise  osmotic  exchanges  surely 
go  on  between  the  vessels  and  the  tissue-cells,  and  the  conditions 
which  determine  the  water  content  of  our  colloid  solutions  constantly 
vary.  The  question  that  remains  is,  do  these  few  factors  account  for 
all  of  the  lymph  formation,  and  are  they  sufficient  by  themselves  to 
explain  the  physiological  regulation  and  the  pathological  variations 
in  the  lymph  flow?  They  are  purely  physical  or  mechanical  causes, 
and  the  "vitalist"  school  will  claim  that  they  are  inadequate  and 
that  "vital  activities"  of  the  cells  play  the  deciding  role.  But  at 
present  the  evidence  that  is  being  accumulated  seems  to  point  more 
and  more  strongly  to  the  conclusion  that  these  "vital  activities" 
are  but  the  result  of  simple  well-known  physical  forces  acting  under 
very  complex  conditions — complex  because  of  the  large  number  of 
different  chemical  compounds  occurring  together,  and  the  varj^ing  in- 
fluence of  circulation,  food  supplies,  cell  structure,  etc. 

ABSORPTION  OF  LYMPH 

By  no  means  all  the  fluid  that  escapes  from  the  vessels,  nor  all  the 
products  of  cell  metabolism,  are  carried  away  in  the  l3'mph — a  con- 
siderable and  perhaps  the  greater  part  of  them  is  absorbed  back 
into  the  capillaries  directly.  A  classical  proof  of  this  is  the  experiment 
of  Magendie,  who  observed  that  if  poisons  were  injected  into  the  leg 
of  an  animal,  which  had  been  separated  from  the  body  entirely  except 
for  the  blood-vessels,  that  poisoning  developed  in  the  usual  manner. 
In  such  experiments  the  lymph-vessels  are  severed  and  probably 
largely  occluded;  hence  it  does  not  solve  the  question  as  to  whether 
substances  are  absorbed  by  the  blood-vessels  under  normal  condi- 
tions. Orlow  found,  however,  that  during  absorption  of  fluid  from  the 
peritoneal  cavity  there  is  no  perceptible  increase  in  the  lymph  flow  from 
the  thoracic  duct.  Addition  of  sodium  fluoride,  a  protoplasmic  poison, 
was  found  to  interfere  with  this  absorption,  for  which  and  other  reasons 
Heidenhain  and  Orlow  considered  that  the  absorj:)tion  doixMuled  upon 
the  "vital  activity"  of  the  cells.  More  nearly  reproducing  normal 
conditions  were  the  experiments  of  Starling  and  Tubby,  who  found 

*°  Hill  ("Recent  Advances  in  Physiology  and  Biochemistry,"  190G,  p.  618)  dis- 
putes the  possibility  of  such  a  thing  as  filtration  prcssiu'c,  on  the  ground  that 
the  structures  within  the  capsule  of  an  organ  must  all  be  under  tlie  influence 
of  the  blood  pressure  alike;  but  with  the  presence  of  an  outlet  for  the  fluid,  as 
in  glands  with  ducts,  filtration  pressure  surely  can  apply. 


ABSORPTION  OF  LYMPH  339 

that  mcthylonc-bluc  or  iiuligo-carniiii(>  injected  into  the  pleura  or 
peritoneum  appeared  in  the  urine  long  before  it  colored  the  lymph  in 
the  thoracic  duct.^^  Adler  and  Mcltzcr  found  evidence,  however, 
that  not  all  the  absorption  is  accomplished  by  the  blood-vessels,  for 
obstruction  of  the  thoracic  duct  retards  absorption.  That  the  ab- 
sorption is  not  dependent  solely  upon  the  circulation  and  blood  pres- 
sure is  shown  by  the  fact  that  absorption  from  the  peritoneal  cavity 
occurs  in  dead  bodies  (Hamburger,  Adler  and  Meltzer). 

The  nature  of  the  mechanism  by  which  fluids  are  taken  into  the 
blood-vessels  is  still  unknown.  We  can  easily  understand  the  en- 
trance of  injected  poisons  and  coloring-matters  from  the  tissues  into 
the  blood,  because  they  are  more  concentrated  at  the  point  of  injection 
than  in  the  blood,  hence  they  may  diffuse  directly  through  the  capil- 
lary wall.  Likewise  we  can  understand  the  diffusion  of  water  from 
a  hypotonic  solution  into  the  blood,  but  how  a  solution  of  the  same 
concentration  as  that  of  the  blood  can  enter  the  blood  is  difficult  to  ex- 
plain. Cohnstein  and  also  Starling  attribute  this  absorption  to  the 
proteins  of  the  blood  in  the  following  manner:  After  a  fluid  is  injected 
into  the  tissues  or  serous  cavities  there  occurs  a  diffusion  exchange 
between  this  fluid  and  the  blood,  until  the  concentration  of  the  crystal- 
loids in  each  is  equal;  but  the  proteins  of  the  blood  cannot  diffuse, 
and  as  they  exert  a  positive  although  very  slight  osmotic  pressure, 
this  difference  in  osmotic  pressure  in  favor  of  the  blood  causes  diffu- 
sion of  the  extravascular  fluid  into  the  blood.  Roth  has  also  applied 
this  idea  in  a  rather  comphcated  manner  to  the  absorption  occurring 
in  metabolic  processes  (see  Meltzer),  but  it  must  be  admitted  that  it  is 
an  unsatisfactory  solution  of  the  problem.  Fischer  would  ascribe  the 
passage  of  fluid  to  the  relative  affinity  of  the  colloids  of  the  blood  and 
of  the  tissues  for  the  fluid,  and  this  would  be  towards  the  blood  when- 
ever the  blood  colloids  had,  from  whatever  possible  cause,  a  greater 
affinity  for  the  fluid  than  the  tissue  colloids. 

Passage  of  the  fluid  from  the  tissues  into  the  lymph  stream  was  very 
easy  to  understand  in  the  light  of  the  older  conception  of  the  lym- 
phatic circulation,  namely,  that  the  lymph-vessels  were  merely  con- 
tinuations of  the  interstitial  spaces;  we  could  then  assume  that  as 
soon  as  the  fluid  left  the  blood-vessels  it  was  practically  within  the 
lymphatic  system,  and  was  crowded  along  the  lymphatic  channels  by 
the  vis  a  tergo,  aided  by  the  valves  of  the  lymph-vessels  and  the  intra- 
thoracic vacuum.  But  it  now  seems,  particularly  through  the  studies 
of  MacCallum,22  that  the  lymphatic  vessels  form  a  closed  system,  not 
in  communication  with  the  interstitial  spaces.  This  being  the  case, 
we  have  to  explain  the  passage  of  the  lymph  through  the  walls  of 
the  lymphatic  vessels,  and  this  is  a  problem  which  is  not  by  any  means 
a  simple  one,  and  which  has  yet  to  be  investigated.     It  is  significant 

21  See  Mendel,  Amer.  Jour.  Physiol.,  1899  (2),  342. 
"  Johns  Hopkins  Hosp.  Bull.,  1903  (14),  105. 


340  ■  EDEMA 

that  the  thoracic  lymph  has  a  higher  osmotic  pressure  than  the  blood 
of  the  same  animal  (Luckhardt),^^  so  that  the  lymph  which  enters 
the  duct  must  do  so  against  the  osmotic  pressure. 

THE  PATHOGENESIS  OF  EDEMA 

With  the  facts  and  h3''potheses  mentioned  in  the  preceding  para- 
graphs in  mind,  we  may  consider  their  bearing  on  the  production  of 
abnormally  large  accumulations  of  lymph  in  the  tissues,  that  is,  edema. 
We  can  imagine  any  one  of  the  following  factors  as  causing  or  helping 
to  cause  such  a  pathological  accumulation: 

1.  Obstruction  to  outflow  through  the  lymph- vessels. 

2.  Increased  blood  pressure. 

3.  Decreased  extravascular  pressure. 

4.  Increased  permeability  of  the  capillary  walls. 

5.  Increased  filterability  of  the  blood  plasma. 

6.  Osmotic  pressure  changes — either  an  extravascular  increase  or 
an  intravascular  decrease. 

7.  Changes  in  the  affinity  of  the  colloids  for  water. 

These  may  be  taken  up  one  by  one,  and  considered  in  relation  to 
their  bearing  upon  the  general  problem  of  edema. 

1.  Obstruction  to  Outflow  through  the  Lymph=vessels. — Be- 
cause of  the  very  abundant  anastomosis  of  the  lymphatic  vessels  it  is 
extremely  difficult  or  impossible  to  cause  any  appreciable  obstruction 
to  the  lymphatic  circulation  by  occlusion  of  lymphatic  trunks  in  the 
limbs  or  organs  of  the  body,  and  in  pathological  conditions  this  possi- 
ble cause  of  edema  is  seldom  actually  observed.  The  chief  instance 
of  edema  from  lymphatic  obstruction  is  observed  after  occlusion  of  the 
thoracic  duct  by  tumors,  tuberculous  processes,  animal  parasites,  or 
thrombosis;  such  occlusion  is  usually  followed  by  rupture  of  the  duct 
or  its  tributaries,  with  the  production  of  chylous  ascites  or  chylothorax, 
and  chyluria.  Filaria  or  their  ova  may  occup.y  so  many  of  the  lymph- 
atic channels  of  an  extremity  (leg)  or  part  (scrotum)  that  the  anasto- 
motic channels  are  thoroughly  blocked,  with  a  resulting  local  edema 
that  in  course  of  time  is  followed  by  the  production  of  inflammatory 
connective  tissue  and  elephantiasis.-^  Chronic  lymphangitis  or  plug- 
ging of  the  lymph  vessels  by  cancer  cells  may  also  result  in  lymphatic 
obstruction  to  such  an  extent  that  chronic  edema  results.  It  would 
seem,  from  Opie's  experiments,-^  that  the  acute  edemas  may  at  times 
depend  upon  lymphatic  obstruction,  for  he  found  that  exi)erimental 
edema  of  the  liver,  produced  by  cantharidin,  seems  to  be  determined 
by  inflammatory  processes  which  occlude  the  sinuses  of  the  l^-mph 
glands  through  which  the  hepatic  l3'niph  passes. 

="  Anicr.  .Jour.  Physiol.,  1910  (25),  345. 

'''  Miiiison,  Allbutt's  System,  1897  (ii),  10S2. 

"  Jour.  Expcr.  Med.,  1912  (16),  831. 


PATIiaCESESIS  OF  EDEMA  341 

Another  way  in  which  i'(hMna  may  \)v  caused  oi-  infhienced  by 
lymphatic  obstruction  is  generally  overlooked,  but  it  is  possibly  of 
great  importance;  namely,  from  pressure  upon  the  lymph  channels  by 
dilated  vessels  in  hyperemia,  or  by  cellular  exudates  and  swollen 
tissues  in  inflammation.  We  see  evidence  of  this  in  the  rapid  absorp- 
tion of  exudates  that  frequentl}-  follows  the  removal  of  but  a  part  of 
the  fluid  in  a  chest  cavity;  apparently  the  decrease  in  pressure  frees 
the  paths  of  absorption  and  permits  them  to  take  up  the  remaining 
fluid.  In  inflammatory  edema  the  lymphatic  obstruction  is  probably 
not  great,  for  Lassar  found  that  the  amount  of  lymph  escaping  from 
an  edematous  extremity  is  much  greater  than  from  a  normal  one; 
but  in  the  case  of  strangulated  hernias  or  other  conditions  in  which 
edema  results  from  circular  constriction,  obstruction  of  the  h^mphatic 
vessels  may  be  a  factor  of  no  mean  importance.  In  general  stasis  the 
increased  pressure  in  the  veins  of  the  neck  may  interfere  with  the 
passage  of  the  fluid  out  of  the  thoracic  duct  into  the  blood. 

There  is  no  difficulty  in  understanding  edema  from  the  above 
causes — it  is  simply  a  passive  congestion  of  the  lymphatic  circulation, 
and  no  chemical  factors  are  involved.  The  nature  of  the  fluid  found  in 
such  forms  of  edema  will  be  discussed  later. 

2.  Increased  Blood  Pressure. — This  takes  us  back  to  the  filtra- 
tion theory  of  lymph  formation,  and  as  it  is  generally  conceded  that 
more  or  less  fluid  escapes  from  the  vessels  by  this  mechanical  process, ^^ 
the  questions  to  be  decided  are:  Can  and  does  increased  blood  pres- 
sure, alone  and  wdthout  other  aiding  factors,  cause  edema?  If  not, 
does  it  play  an  auxiliarj^  part  in  producing  edema,  and  how  important 
a  part  may  this  be?  Many  experiments  have  been  performed  with  the 
object  of  answering  these  questions,  with  more  or  less  conflicting  re- 
sults. Cohnheim  demonstrated  that  vasodilation  (active  hyperemia) 
alone  will  never  bring  on  an  edema;  and  many  observers  state  that 
ligation  of  the  femoral  or  other  large  veins  will  not  cause  edema  in 
animals.  However,  when  the  vein  is  occluded,  and  the  arteries  are 
dilated  by  cutting  their  vasoconstrictor  nerves,  then  edema  may  result 
(Ranvier,  Cohnheim);  but  whenever  venous  outflow  is  impeded,  we 
have  other  factors  than  simply  increased  pressure  to  consider,  for  the 
nourishment  of  the  parts  is  decidedly  impaired,  and,  as  we  shall  see 
later,  this  may  be  of  much  greater  importance  than  is  the  associated 
rise  in  blood  pressure.  To  produce  edema  in  the  lungs  by  mechanical 
forces  it  is  necessary  to  ligate  the  aorta  and  its  branches,  or  the  pul- 
monary veins  (Welch),  As  such  high  pressures  do  not  occur  in  any 
pathological  concHtions,  it  is  safe  to  assume  that  increased  pressure 
alone  is  not  capable  of  causing  by  itself  the  pulmonarj^  edema  so  fre- 
quently observed  clinically.     Welch, ^^  however,  has  supported  the 

^^  A  rise  of  blood  pressure  leads  to  an  increase  in  the  hemoglobin  of  the  blood, 
presumably  because  the  fluid  is  forced  out  into  the  tissue  spaces  (Scott,  Amer. 
Jour.  Physiol.,  1917  (44),  298). 

"  Virchow's  Arch.,  1878  (72),  375;  see  also  Meltzer  {loc.  cit.). 


342  EDEMA 

hypothesis  that  a  disproportion  between  the  working  power  of  the 
left  ventricle  and  of  the  right  ventricle  may  lead  to  pulmonary  edema 
through  pulmonary  hyperemia.  In  the  edema  of  passive  congestion, 
increased  blood  pressure  would  seem  to  be  an  important  factor,  and 
there  is  no  doubt  that  with  an  increased  pressure  of  the  degree  ob- 
served in  such  conditions  some  increase  in  the  hanph  flow  would 
result ;  but  from  the  evidence  at  hand  it  is  improbable  that  the  amount 
of  lymph  so  secreted  would  ever  be  more  than  the  lymph-vessels  could 
carry  away.  Even  the  added  obstruction  to  lymphatic  flow  due  to 
pressure  upon  the  lymph  capillaries  by  congested  blood-vessels,  and 
the  resistance  to  the  lymph  escaping  from  the  thoracic  duct  offered  by 
the  increased  pressure  in  the  subclavian  vein,  would  not  satisfac- 
torily account  for  the  edema  of  cardiac  incompetence.  Not  to  go  into 
details  here,  it  may  be  stated  that  the  impression  prevails  that  uncom- 
plicated rise  in  blood  pressure  is  not  sufficient  by  itself  to  produce 
edema.  Some  of  the  reasons  for  belittling  this  factor  will  be  brought 
out  in  the  subsequent  discussion. 

3.  Decreased  Extravascular  Pressure. — This  factor  is  particu- 
larly prominent  in  the  so-called  "ede^na  ex  vacuo,"  which  occurs  after 
the  absorption  of  an  area  of  tissue  so  located  that  the  surrounding 
tissues  cannot  contract  or  fall  in  to  fill  the  gap,  e.  g.,  brain  softening, 
serous  atrophy  of  fat.  A  still  better  example,  however,  is  the  edema 
that  follows  local  decrease  in  atmospheric  pressure  in  "cupping." 
In  these  instances  the  edema  depends  partly  upon  increased  transu- 
dation, and  partly  on  the  retention  of  the  fluid  in  the  tissues,  because 
it  cannot  well  leave  them  against  the  atmospheric  pressure.  The 
idea  advanced  by  Landerer  that  decreased  elasticity  of  the  tissues  was 
a  possible  cause  of  edema  has  been  attacked  by  Boninger.^^  During 
the  early  stages  of  edema  the  elasticity  of  the  skin  may  be  measurably 
decreased,^^  even  when  no  edema  is  demonstrable  by  palpation,  but 
this  is  not  evidence  that  any  loss  of  elasticity  occupies  a  causative 
relation  to  the  edema.  The  tissues  can  take  up  water  until  as  much  as 
six  kilos  has  been  added  to  the  weight  of  the  entire  body  before  any 
edema  can  be  detected  by  palpation  (Widal).  Edema  ex  vacuo  is 
again  an  illustration  of  edema  due  to  purely  mechanical  causes,  but  it 
is  of  little  practical  importance. 

4.  Increased  Permeability  of  the  Capillary  Walls. — The  im- 
portance of  this  factor  in  the  production  of  edema  was  first  demon- 
strated by  Cohnheim  and  Lichtheim,  who  found  that  the  jiroduction 
of  an  enormous  increase  in  the  amount  of  fluid  in  the  blood  (hydremic 
plethora)  by  injecting  large  quantities  of  salt  solution,  caused  an  edema 
of  the  viscera  and  serous  cavities,  but  not  any  subcutaneous  edema 
until  tlic  skin  had  been  irritated  l)y  some  means,  such  as  hot  water, 
iodiu,  etc.     By  this  irritation  the  capillary  walls  are  injured,  and  an 

2"  Zeit.  exp.  Path.  u.  Ther.,  1905  (1),  163. 
"Schade,  Zeit,.  exp.  Patli.,  1912  (11),  369. 


PATHOGENESIS  OF  EDEMA  343 

excessive  escape  of  the  blood  fluids  follows.  Magnus  also  showed 
that  poisoning  with  arsenic,  which  injured  the  vessels,  favored  the 
experimental  production  of  edema  by  transfusion.  Starling,  as 
noted  before,  observed  that  the  permeability  of  the  capillaries  varies 
normally  in  different  organs  and  tissues,  which  dotorminos  quantita- 
tive and  qualitative  differences  in  the  lymph  normally  flowing  from 
various  vascular  areas.  Heidenhain's  "lymphagogues  of  the  first 
class,  "  which  are  all  poisonous  substances,  probably  act  by  increasing 
the  permeability  of  the  capillaries,  and  in  this  way  they  produce 
local  urticaria,  which  is  often  observed  as  a  result  of  poisoning  by  these 
same  lymphagogues,  e.  g.,  shellfish  and  strawberry  poisoning.  Just 
what  changes  are  produced  in  the  capillary  walls  that  render  them 
more  permeable  we  do  not  know.  Possibly  in  some  instances  it  is  a 
partial  solution  of  the  intercellular  cement  substances,  possibly  an 
enlargement  of  the  stomata  through  loss  of  tonicity  of  the  endothelium 
(Meltzer),  sometimes  it  may  be  actual  death  of  the  endothelial  cells, 
or,  as  Heidenhain  and  Cohnheim  thought,  it  may  be  a  stimulation 
of  the  endothelial  cells  to  increased  secretory  activity.  Fischer  believes 
that  a  change  in  the  hydrophilic  tendency  of  the  colloids,  induced 
especially  by  acids  formed  in  asphyxiated  conditions  of  the  cells,  alter 
their  structure  and  with  that  their  permeability. 

Under  pathological  conditions  increased  permeability  of  the  capil- 
lary walls  is  probably  one  of  the  chief  factors  in  the  production  of 
certain  forms  of  edema.  We  see  evidence  of  it  particularly  in  inflam- 
matory edema,  with  its  protein-rich  exudate.  It  cannot  be  doubted 
that  in  such  conditions  actual  physical  alterations  take  place  in  the 
capillaries,  when  we  see  that  the  slightly  diffusible  proteins  escape 
from  the  vessels  in  the  same  proportions  as  they  exist  in  the  plasma; 
there  can  be  here  no  question  of  heightened  cell  activity  or  increase  in 
osmotic  pressure,  especially  not  when  we  note  the  indistinguishable 
transition  of  such  an  inflammatory  exudate  into  one  containing  leu- 
cocytes and  red  corpuscles,  which  must  pass  through  openings  of 
some  kind  in  the  vessels.  Edema  due  to  inflammation  and  poisoning 
certainly  depends  to  a  large  degree  upon  alterations  in  the  vessel- 
walls.  The  question  remaining  is,  do  edemas  that  are  not  asso- 
ciated  with  distinct  inflammatory  or  toxic  influences  depend  also  upon 
the  vascular  permeability? — does  increased  permeabiHty  ever  lead  to 
the  formation  of  protein-poor  transudates?  Cohnheim  was  inchned 
to  attribute  nearly  all  edema  to  this  cause,  for  in  passive  congestion, 
or  nephritis,  or  any  of  the  common  causes  of  edema,  it  is  easy  to  find 
reason  for  the  belief  that  poisons  may  be  present  in  the  blood;  and 
as  there  was  good  evidence  that  the  blood  pressure  alone  could  not 
account  for  the  edema,  it  was  natural  to  ascribe  all  these  forms  of 
edema  to  the  action  of  toxic  substances  upon  the  capillary  walls,  lead- 
ing to  increased  permeability;  or,  what  might  amount  to  the  same 
thing,  increased  secretory  activity  of  the  endothelium,  as  understood 


344  ,  EDEMA 

"by  Heidenhain.  It  is  impossible  at  this  time  to  eliminate  as  non- 
existent this  secretory-activity  doctrine,  but,  as  we  hope  to  show  later, 
there  exist  other  factors  in  all  these  non-inflammatory  edemas  that 
are  suff'.cient  to  account  for  the  edema  without  our  having  recourse  to 
this  hypothesis.  For  the  present,  therefore,  we  may  consider  altered 
capillary  permeability  as  an  essential  factor  in  edemas  characterized 
by  protein-rich  fluids  (exudates),  and  state  that  the  influence  of  al- 
tered permeability  in  the  production  of  protein-poor  fluids  (trans- 
udates) is  not  proved,  and  is  perhaps  not  of  importance,  although 
the  evidence  of  recent  studies  on  experimental  nephritis  seems  to 
point  more  and  more  to  the  importance  of  vascular  changes  in  acute 
nephritis,  at  least.  ^° 

5.  Increased  Filterability  of  the  Blood  Plasma. — This  takes 
us  back  to  Richard  Bright's  conception  of  renal  drops}'.  He  im- 
agined that  through  the  great  loss  of  albumin  in  the  urine  the  blood 
became  so  thinned  and  w^atery  that  it  could  filter  through  the  vessel- 
walls,  while  normal  plasma,  he  thought,  was  too  thick  and  viscid  to  do 
so.  The  same  idea  was  applied  to  the  edemas  of  cachexia  in  cancer, 
etc.,  chlorosis,  and  all  forms  of  edema  associated  with  a  decrease  in 
the  corpuscular  or  protein  elements  of  the  blood.  With  our  present 
knowledge  of  diffusion  of  crystalloids  and  colloids  we  can  readily  ap- 
preciate that  a  decrease  in  the  blood  colloids,  such  as  might  occur  in 
these  diseases,  could  not  facilitate  the  filtration  of  fluids  through  the 
capillary  walls  to  any  considerable  degree.  On  the  other  hand,  the 
amount  of  colloids  in  the  blood  will  greatl}'  modify'  the  amount  of 
fluid  held  in  the  blood;  e.  g.,  acacia  is  used  in  intravenous  injections 
because  it  holds  in  the  blood  vessels  a  large  amount  of  fluid  by  virtue 
of  its  hydrophilic  character. 

Stewart  and  Bartels  considered  that  in  renal  dropsy  the  increased 
filterability  of  the  plasma  was  not  due  so  nuich  to  the  loss  in  albumin 
as  to  retention  of  water,  which  caused  an  hydremic  plethora.  But 
this  factor  was  soon  eliminated,  for  it  was  found  that  complete  anuria, 
produced  by  ligating  both  ureters,  does  not  cause  edema;  and  also 
that  to  produce  an  edema  by  increasing  the  water  of  the  blood  it  was 
necessary  to  increase  it  many  times  an  nmch  as  it  can  ever  be  increased 
by  disease.  Simply  increasing  the  proportion  of  water  by  removing 
part  of  the  blood  and  injecting  a  corresponding  amount  of  salt  solu- 
tion did  not  cause  edema  (Cohnheim  and  Lichtheim).  We  may,  there- 
fore, look  upon  the  hypothesis  of  increased  filterability  of  the  blood  as 
chiefly  of  historic  interest,  and  not  important  in  the  causation  of 
edema.  In  the  presence  of  other  factors  for  the  production  of  edema, 
however,  the  amount  of  fluid  in  the  vessels  is  important;  thus  Pearce''^ 
found  that  in  experimental  uranium  nephritis  hydremia  exerted  a 
marked  influence  on  the  i)r()du(;ti()n  of  edcMiia. 

30  Sec  Sehmid  and  Schlayer,  Deut.  Arch.  klin.  Med.,  1911  (10-4),  44. 
»'  Arch.  Int..  Med.,  l'»OS  (3),  422. 


PATIKXyENESIS  OF  ICDKMA  345 

().  Disparity  of  Osmotic  Pressure  in  Favor  of  the  Tissues  and 
Lymph  over  the  Blood. — On  a  proccdiiifi;  pa^o  wo  luiv(!  already 
con:sidercd  the  incans  by  which  changes  in  osmotic  pressure  in  the  tis- 
sues are  brought  about,  and  how  they  may  lead  to  an  accumulation 
of  fluid.  The  importance  of  osmotic  pressure  in  causing  pathological 
edema  was  suggested  by  J.  Loeb''-  in  his  studies  on  the  physiological 
action  of  ions.  He  stated  that  edema  occurred  when  the  osmotic 
pressure  was  higher  in  the  tissues  than  it  was  in  the  blood  and  lymph, 
and  the  cause  was  to  be  sought  in  conditions  that  lowered  the  osmotic 
pressure  of  the  blood  and  lymph  or  raised  that  of  the  tissues.  This 
condition  he  found  in  the  accumulation  of  metabolic  products: — in 
the  case  of  muscle,  tetanization  of  a  frog's  muscle  for  ten  minutes 
raised  the  osmotic  pressure  over  one  atmosphere;  separating  a  muscle 
from  its  blood-supply  led  to  such  an  increase  in  osmotic  pressure  that  it 
took  up  water  from  a  4.9  per  cent.  NaCl  solution,  which  has  a  pres- 
sure of  over  thirty  atmospheres.  When  we  consider  that  in  his  studies 
on  lung  edema  Welch  was  able  by  ligation  of  the  aorta  to  raise  the 
blood  pressure  less  than  ifo  atmosphere,  we  begin  to  appreciate  how 
much  more  powerful  are  the  physico-chemical  forces  that  are  at  work 
in  the  body  than  is  the  blood  pressure,  even  of  the  aorta  itself. 

Loeb  found  that  whenever  oxidation  is  impaired  in  a  tissue  its 
osmotic  pressure  rises,  which  he  ascribed  to  the  accumulation  of  in- 
completely oxidized  metabolic  products,  particularly  acids,  and  as  a 
result  the  muscle  takes  up  water  and  becomes  edematous.  On  this 
basis  we  might  explain  the  edema  of  venous  stagnation  as  due  to  ac- 
cumulation of  products  of  metabolism,  partly  because  of  impaired 
oxidation,  partly,  perhaps,  because  of  their  slow  removal  in  the  blood 
on  account  of  the  circulatory  disturbance.  The  so-called  "neurotic" 
edemas  may  possibly  be  explained  by  local  increase  in  metabolic  ac- 
tivity brought  about  by  nervous  stimuli,  which  causes  increased  forma- 
tion of  substances  raising  osmotic  pressure  in  the  stimulated  tissues. 
In  renal  edema  the  retention  of  water  also  seems  to  depend  rather  on 
osmotic  pressure  than  on  circulatory  disturbances  or  alterations  in  the 
vessel-walls,  for  it  has  been  shown  that  retention  of  chlorides,  which 
the  diseased  kidneys  do  not  eUminate  normally,  is  an  important  cause 
of  the  dropsy  in  some  cases.  The  chlorides  accumulating  in  the  tissues 
lead  to  an  increased  osmotic  pressure,  which  causes  the  abstraction  of 
water  from  the  blood  and  its  retention  in  the  tissues.  (The  details  of 
this  subject  will  be  considered  later.)  Conversely,  Meltzer  and  Salant 
found  that  salt  solution  is  absorbed  from  the  peritoneal  cavity  more 
rapidly  in  nephrectomized  rabbits  than  in  normal  rabbits  because 
metabolic  products  accumulate  in  the  blood  and  raise  its  osmotic  pres- 
sure above  normal;  and  it  was  observed  by  Fleisher  and  L.  Loeb^^ 
that  the  rate  of  absorption  of  fluid  from  the  peritoneal  cavity  is  in- 
creased when  the  osmotic  pressure  of  the  blood  is  raised. 

32  Pfluger's  Arch.,  1898  (71),  457. 
"  Jour.  Exper.  Med.,  1910  (12),  510. 


346  EDEMA 

There  are  3ome  difficulties,  however,  in  applying  the  influence  of 
osmotic  pressure  as  an  explanation  of  all  edemas.  For  example,  in 
edema  of  the  lungs,  as  Meltzer  points  out,  what  is  the  force  that  drives 
the  fluid  into  the  empty  air-cells?  Equally  difficult  to  explain  as  the 
result  of  osmotic  disturbance  is  the  distribution  of  fluid  that  is  seen 
in  cardiac  dropsy.  The  fluid  does  not  accumulate  in  the  tissues  where 
metabolism  is  greatest,  or  where  the  most  oxygen  is  used;  but  rather 
in  the  inactive  subcutaneous  tissues  and  in  the  serous  cavities.  Possi- 
bly the  original  transudation  does  occur  in  the  muscles  and  sohd 
viscera,  and  the  fluid  is  then  mechanically  forced  out  of  them  into 
the  surrounding  tissue-spaces,  later  settling  according  to  the  laws  of 
gravity  or  according  to  the  distensibility  of  the  tissues.  It  is  im- 
portant to  take  into  consideration  the  fact  that  demonstrable  edema 
does  not  manifest  itself  until  a  very  large  quantity  of  fluid  has  been 
retained  by  the  body — as  much  as  six  kilos,  according  to  Widal. 

Increased  Hydration  Capacity  of  the  Tissue  Colloids. — According 
to  Fischer's  theory  this  factor  is  of  greater  importance  than  any  of  the 
preceding,  and  of  chief  importance  in  increasing  the  amount  of  water 
present  in  the  tissues  are  organic  acids  formed  during  metabolism. 
For  example,  the  great  power  of  asphyxiated  muscle  to  take  up  water 
from  a  strong  salt  solution,  which  J.  Loeb  ascribed  to  the  osmotic 
pressure  of  the  acids  formed  in  asphyxia,  is  attributed  by  Fischer 
to  the  influence  of  these  acids  upon  the  capacity  of  the  colloids  for 
water,  and  this  explanation  seems  to  be  in  better  agreement  with  the 
facts,  especially  since  Overton  has  shown  that  even  if  all  the  proteins, 
carbohydrates  and  fats  in  a  muscle  were  split  into  the  greatest  possi- 
ble number  of  simple  molecules  and  ions,  the  resulting  osmotic  pres- 
sure would  not  be  sufficient  to  account  for  the  amount  of  water  taken 
up.  Furthermore,  when  cells  with  demonstrable  semi-permeability 
die,  they  at  once  lose  their  semi-permeability,  and  in  consequence  their 
osmotic  pressure  falls — but  dead  cells  and  tissues  often  exhibit  great 
power  of  taking  up  water  and  becoming  edematous.^'*  It  is  an  in- 
disputable fact  that  edema  is  especially  associated  with  conditions  of 
asphj^xiation,  and  the  attempt  to  explain  this  by  the  increased  osmotic 
pressure  of  the  products  of  incomplete  oxidation  seem  to  harmonize 
with  the  facts  far  less  successfully  than  the  apphcation  of  the  prin- 
ciple of  colloidal  swelling.  A  common  error  of  the  critics  of  this 
theory  is  that  of  assuming  that  free  acid  must  be  present  to  cause 
swelling.  This  is  not  at  all  true.  An  amount  of  acid  far  less  than 
enough  to  saturate  the  acid-binding  property  of  a  protein  or  to  be 
detected  by  indicators  will  greatly  increase  the  amount  of  water  which 

"  The  secreted  fluid  of  postmortem  thoracic  lymph  flow  diff"ors  from  normal 
thoracic  lymph  in  bein{;  more  cloudy,  often  bloody,  contains  more  solids,  has  a 
higher  molecular  concentration  with  decreased  electrical  conductivity  (.lappelli 
and  d'Errico,  Zeit.  f.  Biol.,  1907  (50)  1),  all  of  which  findings  are  in  agreement 
with  the  hypothesis  that  postmortem  lymph  flow  depends  upon  changes  in  the 
cells,  caused  oy  asphyxia  and  not  dissimilar  to  the  changes  of  acute  nephritis. 


PATHOGENESIS  OF  EDEMA  347 

this  protein  will  combine.  Presumably  the  colloidal  carbohydrates 
and  lipoids  may  also  play  a  part  in  the  water  absorption  of  tisr^ues. 

Fischer's  theory  of  edema,  in  his  own  words,  is  this:  "A  state  of 
edema  is  induced  whenever,  in  the  presence  of  an  adequate  supply 
of  water,  the  affinity  of  the  colloids  of  the  tissues  for  water  is  increased 
above  that  which  we  are  pleased  to  call  normal.  The  accumulation 
of  acids  within  the  tissues  brought  about  either  through  their  abnor- 
mal production,  or  through  the  inadequate  removal  of  such  as  some 
consider  normally  produced  in  the  tissues,  is  chiefly  responsible  for 
this  increase  in  the  affinity  of  the  colloids  for  water,  though  the  possi- 
bility of  explaining  at  least  some  of  the  increased  affinity  for  water 
through  the  production  or  accumulation  of  substances  which  affect 
the  colloids  in  a  way  similar  to  acids,  or  through  the  conversion  of 
colloids  which  have  but  little  affinity  for  water  into  such  as  have  a 
greater  affinity,  must  also  be  borne  in  mind."  In  support  of  this 
theory  he  advances  evidence  which  he  interprets  as  indicating  that: 
(i)  "An  abnormal  production  or  accumulation  of  acids,  or  condi- 
tions predisposing  thereto,  exist  in  all  states  in  which  we  encounter 
the  development  of  an  edema.  (2)  The  development  of  an  edema  in 
tissues  is  antagonized  by  the  same  substances  which  decrease  the 
affinity  of  the  (h>  drophilic)  emulsion  colloids  for  water  (salts)  and  is 
unaffected  by  uhe  presence  of  substances  which  do  not  do  this  (non- 
electrolytes).  (3)  Any  chemical  means  by  which  we  render  possible 
the  abnormal  production  or  accumulation  of  acids  in  the  tissues  is 
accompanied  by  an  edema." 

There  are  many  features  of  lymph  formation  and  edema  with 
which  this  theory  seems  to  harmonize  well,  and  others  with  which  it 
does  not  seem  to  agree  so  well,  if  at  all,  so  that  at  this  time  it  is  a 
fair  statement  that  the  theorj'  is  under  consideration,  but  the  limita- 
tions of  its  applicability  have  not  yet  been  agreed  upon.  It  has  met 
with  much  adverse  criticism,  some  of  which  was  poorly  founded,  but 
the  fact  cannot  be  disputed  that  the  amount  of  water  that  colloids 
will  hold  varies  greatly  with  changes  in  the  colloids.  We  may  not 
know  absolute^,  at  present,  whether  the  changes  that  take  place  in 
the  colloids  during  life  are  great  enough  to  alter  their  water  content 
appreciably,  but  it  is  highly  probable  that  they  are.  In  many  in- 
stances the  principles  of  colloidal  hydration  offer  the  best  explanation 
of  observed  conditions,  and  their  application  often  elucidates  matters 
more  satisfactorily^  than  any  other  working  hj'pothesis.  Certainly 
the}'  cannot  be  disregarded  in  considering  the  factors  that  may  come 
into  play  in  producing  edema. 

Summary. — We  find  that  a  number  of  factors  may  be  considered 
as  responsible  for  edema,  some  of  them  being  prominent  in  one  in- 
stance, some  in  another,  but  in  few  cases  can  we  consider  one  factor 
alone  as  the  sole  cause.  In  most  of  the  forms  of  edema,  such  as  those 
due  to  renal  disease  and  cardiac  disease,  it  now  seems  probable  that 


348  EDEMA 

either  osmotic  pressure  changes  or  changes  in  the  affinity  of  the  tissue 
colloids  for  water,  play  the  most  important  part;  whereas  in  inflamma- 
tory edema  there  can  be  no  question  that  alteration  in  the  capillary 
walls  is  the  most  essential  factor.  But  the  mechanical  factor  of  blood 
pressure  cannot  be  disregarded,  although  by  itself  seldom  sufficient  to 
cause  edema;  associated  with  other  factors  it  is  undoubtedly  an  im- 
portant agency,  for  there  are  few  edemas  that  arc  not  associated  with 
increased  blood  pressure.  Hydremia  and  hydremic  plethora  may  al- 
most be  disregarded,  except  in  so  far  as  they  may  cause  altered  metab- 
olism in  the  tissues,  injury  to  vessel-walls,  over-saturation  of  the  blood 
colloids,  and  decreased  osmotic  pressure  within  the  vessels.  Ljanph- 
atic  obstruction  is  possibly  a  factor  of  some  secondarj^  importance  if 
we  consider  that  distended  vessels  and  tense  tissues  may  occlude  the 
lymph  capillaries. 

Special  Causes  of  Edema 

We  may  now  consider  which  of  the  above  factors  are  at  work  in 
bringing  about  edema  under  the  conditions  in  which  it  is  usually 
observed  clinically.  Before  taking  up  the  detailed  consideration  of 
edematous  conditions,  however,  it  may  be  well  to  call  attention  to  the 
fact  that  our  knowledge  of  edema,  and  especially  its  clinical  recog- 
nition and  study,  has  been  handicapped  by  the  lack  of  a  suitable  ob- 
jective method  of  detecting  and  measuring  edema.  We  are  in  the 
same  position  in  respect  to  edema  that  we  were  to  blood  pressure 
when  the  only  measure  was  the  clinician's  forefinger.  An  attempt 
to  remedy  this  defect  has  been  made  by  Schade,^^  whose  ''elastometer" 
reveals  and  measures  degrees  of  edema  not  discernible  bj^  the  palpating 
finger.  A  study  of  edema  with  this  instrument  in  the  hands  of 
Schwartz^^  has  revealed  many  interesting  facts,  but  as  yet  the  appa- 
ratus is  too  complicated  for  general  clinical  use. 

"Cardiac"  Edema. — Passive  congestion  introduces  nearly  all 
these  aforementioned  factors,  for  in  addition  to  the  increased  blood 
pressure  there  is  also  an  opportunity  for  changes  in  the  capillary  wall, 
either  from  stretching  and  thinning  of  the  cells  and  cement  substances, 
or  from  "loss  of  tone"  in  the  endothelium  surrounding  the  stomata 
(Meltzer),  or  from  toxic  injury  by  accumulated  products  of  tissue 
metabolism.  When  the  stasis  is  nearly  complete,  or  if  it  is  comp'ete 
for  a  time  and  then  relieved,  the  endothelium  may  })e  injured  through 
lack  of  nourishment.  As  the  edematous  fluid  in  chronic  passive  con- 
gestion is  usually  of  a  watery  type,  poor  in  proteins,  the  edema  is 
probably  less  dependent  upon  capillary  permeability  than  upon  other 
factors,  except  in  the  ease  of  acute  stasis,  when  the  fiuid  partakes  of 
the  character  of  the  exudates.  Presumably  the  accunuilation  of 
crystalloids  within  the  tissues  also  plays  a  part  in  this  form  of  edema, 
as  the  osmotic  pressure  is  raised  in  tissues  having  deficient  oxygen 

"  Arch.  Int.  Med.,  1910  (17),  390  and  4r-,9. 


NEPHRITIC  EDEMA  349 

supply.  But  Fischer  liolds  tluit  tl)c  retluction  in  oxidation  acts  chiefly 
by  increased  production  of  acids,  which  greatly  increase  the  affinity 
of  the  tissue  colloids  for  water  and  at  the  same  time  alter  the  colloidal 
state  of  the  capillary  endothelium  so  that  the  capillaries  ijecome  more 
permeable.  Finally,  there  is  probably  more  or  less  obstruction  to 
lymphatic  outflow  because  of  the  increased  pressure  on  the  lymphacic 
channels,  and  perhaps,  also,  in  the  case  of  cardiac  incompetence,  ob- 
struction to  the  discharge  of  lymph  from  the  thoracic  duct  into  the 
subchu'ian  vein  against  the  high  intravenous  pressure. 

Renal  Edema. — We  must  recognize  under  this  heading  two  dif- 
ferent types  of  edema.  ■  In  acute  nephritis  (e.  g.,  in  scarlatina)  toxic 
materials  appear  to  be  the  chief  cause,  and,  as  Senator  contends,  in- 
jure alike  the  capillaries  of  the  renal  glomerules  and  of  the  subcu- 
taneous tissues;  in  each  case  there  results  an  increased  permeability 
which  is  manifested  by  albuminuria  as  a  result  of  the  injury  to  the 
renal  capillaries,  and  by  edema  as  a  result  of  the  injury  to  the  tissue 
capillaries.  This  sort  of  edema  is  allied  to  that  produced  by  peptone 
and  similar  13'mphagogues,  and  we  might  well  imagine  that  the  mech- 
anism consisted  merely  in  an  injur}-  to  the  capillaries  through  which 
excessive  fluid  is  driven  by  the  blood  pressure,  were  it  not  for  such 
observations  as  those  of  ]\Iendel  and  Hooker,^*'  who  found  that  post- 
mortem flow  is  increased  b}-  these  h'mphagogues  also.  We  can 
hardly  account  for  the  force  exhibited  in  postmortem  lymph  flow  on 
any  other  ground  than  that  it  is  furnished  by  osmotic  pressure  or 
colloidal  absorption  unless  we  wish  to  fall  back  upon  "vital  activity" 
of  the  surviving  cells.  Hence  it  is  probable  that  even  in  the  edemas 
of  toxic  conditions,  such  as  acute  nephritis,  physico-chemical  factors 
play  a  part,  the  responsible  substances  probabl}''  being  abnormal  or 
excessive  metabolic  products  of  the  cells  affected  by  the  poisons.  An 
interesting  observation  made  by  Bence"  is  that  nephrectomized  rabbits 
develop  an  edema  even  when  they  are  given  no  water  at  all;  this  would 
seem  to  indicate  an  increased  affinity  of  the  tissues  for  water  when 
the  renal  functions  are  deficient.  Hydremia  is  always  a  favoring 
factor,  however,  and  probably  important  in  nephritic  edema, ^*  while 
nearly  all  students  of  acute  experimental  nephritis  find  evidence  that 
the  resulting  edema  depends  very  much  upon  the  changes  in  the  vessel- 
walls.39 

In  the  more  common  edema  of  chronic  nephritis  we  have  to  con- 
sider, among  other  factors,  the  blood  pressure.  That  this  is  not  an 
essential  or  even  important  cause,  however,  is  shown  by  the  fact 
that  edema  is  usually  much,  less  marked  in  interstitial  nephritis  with 
high  blood  pressure  than  it  is  in  parenchymatous  nephritis  with  a 

3«  Amer.  Jour,  of  Physiol.,  1902  (7),  380. 
"  Zeit.  f.  klin.  Med.,  1909  (67),  69. 

38  1  earce,  Arch.  Int.  .Med.,  1909  (3),  422. 

39  See  Schmidt  and  Schlaver,  Deut.  Arch.  klin.  Med.,  1911  (104),  44;  Pollak 
Wien.  kUn.  Woch.,  1914  (27),  98. 


350  EDEMA 

« 

much  lower  pressure.  Toxic  substances  are,  of  course,  also  present  in 
the  blood,  and  may  alter  capillary  permeabihty;  these  toxic  substances 
may  account  for  the  lo-calized  edemas  and  erythemas  sometimes  ob- 
served in  nephritis.  But  probably  most  important  is  the  action  of 
the  crystalloids  which  the  kidney  does  not  excrete,  and  which  seem 
to  be  stored  up  in  the  tissues,  where  they  cause  transudation  of  water 
under  the  influence  of  their  osmotic  pressure.  For  example,  Rzent- 
kowski"*"  found  that  the  average  lowering  of  the  freezing-point  by  the 
edematous  fluid  in  nephritis  was  0.583°,  in  cardiac  dropsy  it  was  0.548°, 
and  in  tuberculous  pleuritis  0.526°.  This  indicates  ':hat  the  osmotic 
concentration  of  the  fluid  is  highest  in  renal  dropsy,  and  supports  the 
belief  that  here  and  in  cardiac  dropsy  osmotic  pressure  plays  a  more 
important  part  than  it  does  in  inflammatory  exudation. ^^  Of  the 
crystalloids  that  cause  accumulation  of  fluid  in  the  tissues,  sodium 
chloride  seems  to  be  the  most  important. 

Retention  of  Chlorides  in  Edema. ^^ — From  the  investigations  made  by  numer- 
ous clinicians,  especially  the  French,  it  appears  that — (1)  in  nephritis  with  edema 
a  retention  of  sodium  chloride  frequently  occurs;  (2)  that  elimination  of  the 
chlorides  is  often  increased  during  periods  of  improvement  of  the  edema;  (3) 
that  a  reduction  of  the  amount  of  chlorides  in  the  diet  sometimes  causes  a  great 
improvement  in  the  edema,  while  administration  of  chlorides  may  make  the 
edema  much  worse.  There  are,  however,  observations  that  also  indicate  that 
chloride  retention  does  not  account  for  many  cases  of  renal  drops}',  for  com- 
monly the  above-mentioned  conditions  are  not  fulfilled."  Nevertheless,  it  cannot 
be  denied  that  chloride  retention  is  sometimes  an  important  causative  factor  in 
the  edema  of  parenchymatous  nephritis."  If  the  retained  chlorides  obeyed  the 
ordinary  laws  of  diffusion,  we  should  expect  them  to  become  distributed  alike  in 
the  blood  and  tissues,  so  that  they  would  merely  cause  an  equal  increase  in  the 
fluids  of  the  blood  and  of  the  tissues;  that  is  to  say,  there  would  be  an  hydremic 
plethora  due  to  retention  of  water  in  the  body  by  the  accumulating  chlorides. 
But,  according  to  a  number  of  observers,  there  is  a  specific  retention  in  the  tissues, 
which  Strauss  calls  " historetention,"  and  which  explains  the  local  edema.  The 
way  in  which  the  historetention  is  produced  is,  however,  not  understood,  and 
not  all  observers  accept  this  hypothesis.  If  chlorides  do  bear  a  causative  rela- 
tion to  edema,  the  predilection  of  the  subcutaneous  tissues  for  edematous  accumu- 
lations may  be  explained  by  the  observation  that  when  salt  is  given  to  an  animal 
an  undue  proportion  (28-77  per  cent.)  accumulates  in  the  skin."  In  many 
conditions  other  than  nephritis,  there  is  also  a  chloride  retention  (e.  g.,  pneu- 
monia, cardiac  incompetence,  sepsis,  typhoid),  and  the  edemas  observed  in  these 
diseases  may  possibly  depend  upon  chloride  retention,  as  many  French  authors 

^0  Berl.  klin.  Woch.,  1904  (41),  227. 

^1  Epstein  (Amer.  Jour.  Med.  Sci.,  1917  (154),  638)  calls  attention  to  the  de- 
crease of  serum  proteins  (sometimes  GO  to  70  per  cent.)  and  ascribes  the  edema  to 
lowered  osmotic  pressure  of  the  blood  from  loss  of  colloids.  Low  protein  content 
of  the  blood  might  more  probably  favor  edema  by  reducing  the  amount  of  fluid 
which  the  blood  can  hold  as  a  hvdrophile  colloid. 

'2  Literature,  r6sum6  by  Widal  and  Javal,  Jour.  Physiol,  et  Pathol,  1903  (5), 
1107  and  1123;  Rumpf,  Miinch.  med.  Woch.,  1905  (52),  393.  Review  in  Albu 
and  Neuberg's  "Mineralstoffwochsel,"  Berlin,  1900,  pp.  171-178;  Georgopulus, 
Zeit.  klin.  Med.,  1900  (60),  411;  Christian,  Boston  Med.  and  Surg.  Jour.,  1908 
(158),  416;  Palmer,  Arch.  Int.  Med.,  1915  (15),  329. 

"See  Blooker,  Deut.  Arch.  klin.  Med.,  1909  (96),  80;  Fischer,  "(Edema  and 
Nephritic." 

**  See  Borchardt,  Deut.  med.  Woch.,  1912  (38),  1723. 

"  Schade,  Zeit.  exp.  Path.  u.  Ther.  1913  (14),  1.  Also  gives  an  interesting 
discussion  of  the  relation  of  the  skin  to  euenui. 


INFLAMMATORY  EDEMA  351 

supRest.  Kunijif,  indeed,  often  found  more  chlorides  in  edematous  fluids  of  non- 
nephritic  origin  than  in  nephritic  edema.'*'  Fischer  holds  that  the  retention 
of  chlorides  in  edema  is  secondary  and  not  primary,  for  he  found  that  tissues 
made  to  take  up  more  water  through  acidification,  also  take  up  an  increased 
amount  of  chlorides. 

Inflammatory  Edema. — Although  here  the  alterations  in  the  cap- 
illary walls  play  an  essential  role,  as  shown  by  the  protein-rich  na- 
ture of  the  exudates,  yet  most  of  the  other  factors  are  added.  In- 
creased blood  pressure  is  prominent;  lymph  outflow  is  impeded  by 
plugging  of  the  lymphatic  channels  by  clots  and  leucocytes,  and  by 
pressure  on  the  outside;  there  is,  undoubtedly,  an  excessive  forma- 
tion of  metabolic  products  in  the  tissues,  to  cause  exosmosis,  and  the 
asphyxial  conditions  in  inflamed  tissues  favor  acid  formation  which 
ma}'  cause  in  the  colloids  an  increased  affinity  for  water.  According 
to  Oswald'*^  the  permeability  of  the  vessels  for  proteins  becomes  spe- 
cifically altered  in  inflammation,  so  that  not  only  the  less  viscous 
albumin  and  pseudoglo])uhn  pass  through  their  walls,  but  also  the  more 
viscous  euglobulin  and  fibrinogen.  To  this  class  of  edemas  belong 
also  the  urticarias  which  follow  the  ingestion  of  various  toxic  sub- 
stances, many  of  which  can  be  shown  experimentally  to  be  lympha- 
gogues.  A  good  example  is  the  urticaria  which  often  follows  the 
injection  of  antitoxic  or  other  foreign  serums,  particularly  their  re- 
peated injection;  in  experimental  animals  such  a  serum  may  cause 
death  very  quickly  by  acute  pulmonary  edema.  All  these  poisons 
probably  produce  urticarial  edema  by  injury  to  the  capillary  walls  in 
the  subcutaneous  tissues,  and  possibly  changes  in  the  hydrophilic 
properties  of  the  tissue  colloids  are  also  produced  by  the  poisons.  In 
the  action  of  vesicants  especially,  it  may  well  be  questioned  if  changes 
in  the  capillary  walls  and  active  hyperemia  are  not  supplemented  by 
local  metabolic  alterations.  The  edema  which  follows  the  sting  of 
insects,  which  are  known  to  secrete  into  the  wound  such  acids  as 
formic,  seems  to  be  a  particularly  good  illustration  of  the  production 
of  edema  by  the  influence  of  acids  on  the  tissues  (Fischer). 

Neuropathic  Edema. — Until  we  understand  better  than  we  now 
do  the  manner  in  which  nervous  impulses  modify  metabolism,  it  will 
be  difficult  to  estimate  properly  the  importance  of  nervous  impulses 
in  the  production  of  edema.  That  nervous  control  is  a  possible  factor 
is  well  shown  by  manj^  experiments;  for  example,  simple  ligation  of 
the  femoral  vein  in  animals  does  not  cause  edema,  but  if  the  sciatic 
nerve  is  cut  the  vasoconstrictors  are  paralyzed,  and  edema  mav  follow 
(Ranvier).^^  In  this  case  the  nervous  influence  is  only  indirect 
through  its  vasomotor  effects.     Similarly,  stimulation  of  vasodilator 

*^  Breitmann  (Zentr.  inn.  Med.,  1913  (34),  633)  describes  under  the  name  of 
"soda  dropsy"  a  form  of  edema  which  results  from  excessive  administration  of 
sodium  bicarbonate  to  correct  acidosis  in  diabetes. 

"  A.  Oswald,  Zeit.  exp.  Path.,  1910  (8),  226. 

*^  Similarly,  pulmonary  edema  follows  experimental  hydremia  onlj-  when  the 
vagi  are  cut  (F.  Kraus,  Zeit.  exp.  Path.,  1913  (14),  402). 


352  EDEMA 

fibers  may  cause  edema.  It  is  furthermore  possible  that  nervous 
stimulation  may  lead  to  excessive  metabolic  activity,  with  an  ac- 
cumulation of  crystalloidal  products  and  acids  sufficient  to  cause 
edema  when  supplemented  by  active  congestion  and  some  resulting 
pressure  upon  the  lymph-vessels.  There  are  certainly  many  instances 
in  which  edema  seems  to  depend  upon  nervous  disturbance;  for  ex- 
ample, edema  in  the  area  of  distribution  of  a  neuralgic  nerve;  sudden 
joint  effusions  in  tabetic  arthropathy;  and  especially  the  typical 
"angioneurotic"  edema. ^^  The  only  explanation  that  seems  open  is 
the  one  given  above,  namely,  a  combination  of  local  h^-peremia  and  in- 
creased metabolic  activity.  Even  the  urticarias  of  apparently  me- 
chanical origin  (urticaria  factitia),  show  evidence  of  a  toxic  action, 
in  that  there  occurs  a  severe  nuclear  fragmentation  (Gilchrist).^" 

Hereditary  Edema. — In  a  number  of  families  there  has  been  ob- 
served a  peculiar  inherited  tendency  to  the  occurrence  of  acute  attacks 
of  local  edema,  which  not  infrequently  have  proved  fatal  when  in- 
volving the  glottis. ^^  There  can  be  little  question  that  these  instances 
of  hereditary  edema  depend  upon  a  nervous  affection  of  some  kind,  it 
being  practically  an  angioneurotic  edema;  but  how  the  edema  is  pro- 
duced, and  what  the  nature  of  the  nervous  alteration  may  be,  are  as 
mysterious  as  are  most  other  so-called  "nervous  inheritances."  There 
also  are  cases  of  congenital  edema,  which  may  occur  repeatedly  in 
the  fetuses  of  the  same  mother  and  cause  habitual  miscarriage;^^  and 
still  another  class  of  cases  in  which  the  children  are  born  apparently 
healthy,  but  develop  fatal  dropsy  w^hen  a  few  weeks  old.^''  Nothing 
is  known  as  to  the  cause  of  this  condition.  Patein*^  has  analyzed  the 
fluid  in  a  case  of  congenital  ascites  and  found  it  somewhat  more  like 
an  exudate  than  a  transudate. 

Nutritional  Edema  ("War  Dropsy"  or  Famine  Edema). ^(Discussed 
under  Deficiency  Diseases,  Chapter  xii.) 

COMPOSITION   OF  EDEMATOUS  FLUIDS^^ 

As  is  well  known,  the  composition  of  edematous  fluids  varies  greatly 
according  to  the  cause  of  the  edema  and  the  place  where  it  occui's. 
In  general,  non-inflammatory  edemas  (transudates)  contain  much  less 
protein  than  do  the  inflammatory  exudates,  as  is  shown  by  the  follow- 
ing taljle  of  analyses  by  Halliburton^'*  and  by  Rornheim's-'"  deter- 
minations of  proteins  in  ascitic  fluids. 

'"'  Metabolism  in  angioneurotic  edema  is  discussed  bv  ISIilliMand  Pepper,  Arch. 
Int.  Med.,  191G  (18),  551. 

'-"Johns  Hopkins  Hosp.  Bull.,  1908  (19),  49. 

"Literature,  see  Fairbanks,  Amer.  Jour.  Med.  Sci.,  1904  (127),  877;  Hope 
and  French,  Quart.  Jour.  Med.,  1908  (1),  312;  Crowder,  Arch.  Int.  Med.,  1917  (20), 
840. 

"  W.  Fischer,  Berl.  klin.  Woch.,  1912  (49),  2403. 

"Edgeworth,  Lancet,  1911  (181),  211). 

"Jour.  IMiariu.  et  Cliim.,  1910  (102),  209. 

"  Many  data  are  given  by  Cierliarlz,  ilamlbuoh  dor  Bioi'heniio,  190S,  II  (2),  137. 

'-0  Adaiiii,  Allbutt's  System,  189(5  (1),  97. 

"  (Quoted  by  llamnuirsten,  "Physiological  C'homistrj-." 


COMPOSITION  OF  EFFUSIONS 
Table  I 


353 


Parts  per  100  ot  fluid 


Sp.  gr. 


Total 
protein 


Fibrin 


Serum- 
globulin 


Serum- 
albumin 


Acute  pleurisy . . . 
Acute  pleurisy. . . 
Acute  pleurisy . . . 

Hydrothorax  "1 
Aver,  of  3  cases  / 


1.023 
1.020 
1.020 


5.123 

3.4371 

5.2018 


0.016 

0.0171 

0.1088 


3.002 

1 . 2406 
1.76 


2.114 

1.1895 

3.330 


1.014 


1.7748      0.0086 


0.6137 


1.1557 


Table 

II 

Ascitic  fluid  in 

Parts  of  protein  to  1000 

c.c.  fluid 

Max. 

Min. 

Mean 

Cirrhosis  of  the  liver 

34.5 
16.11 

5.6 
10.10 

9  69-21  06 

Bright 's  disease 

15.6-10.36 

Tuberculous  and  idiopathic 
Carcinomatous  peritonitis. . 

peritonitis.  .  .  . 

55.8 

5 1  .  20 

18.72 
27.00 

30.7-37.95 
35 . 1-58 . 96 

The  specific  gravity  varies  nearly  in  direct  proportion  to  the  amount 
of  proteins,  that  of  transudates  usuall}^  being  below  1.015,  and  exu- 
dates above  1.018,  although  there  are  many  exceptions.  Indeed,  it  is 
often  very  difficult  to  decide  whether  a  given  fluid  is  an  exudate 
or  a  transudate. ^^  According  to  Rzentkowski,^^  the  transudates  at 
the  moment  they  pass  out  of  the  vessels  are  simply  solutions  of  crystal- 
loids in  water  and  quite  free  from  protein;  the  small  amount  of  protein 
found  in  transudates  he  ascribes  to  protein  pre-existing  in  the  tissue- 
spaces.  This  idea  is  hardly  acceptable  in  view  of  the  known  per- 
meability of  the  vessel-walls  for  proteins  in  normal  conditions;  more 
probably  in  cardiac  and  renal  dropsies  the  quantity  of  protein  escap- 
ing from  the  vessels  is  not  greatly  different  from  normal,  but  the 

68Rivalta  (Rif.  Med.,  1903;  Biochem.  Centr.,  1904  (2),  529)  has  suggested 
the  following  test  to  distinguish  exudates  and  transudates:  Into  a  beaker  con- 
taining 200  c.c.  of  water  with  4  drops  of  glacial  acetic  acid,  let  fall  a  few  drops 
of  the  fluid  to  be  tested.  If  an  exudate,  a  bluish-white  line  is  left  transiently 
behind  the  sinking  drops,  due  to  precipitation  of  the  euglobulin  and  fibrinogen. 
This  test,  and  also  certain  modifications  (see  Rivalta,  Policlinico,  1910  (17), 
676),  seem  to  give  quite  reliable  results.  (See  Ujihard,  Berl.  klin.  Woch.,  i914 
(51),  1112).  With  tuberculous  effusions  Rivalta's  test  is  positive,  but  not  Mo- 
relli's  test,  which  consists  in  dropping  the  fluid  into  saturated  HgClo  solution,  a 
yellowish  ring  of  albuminate  forming  with  non-tuberculous  exudates,  and  a  gran- 
ular precipitate  with  transudates.  (See  Zannini,  Gaz.  degli  Osped.,  1914  (4), 
461).  Memmi  (Clin.  Med.  Ital.,  1905,  No.  3)  suggests  the  larger  content  of 
lipase  as  a  means  of  distinction  of  exudates.  Tedeschi  (Gaz.  degli  Osped.,  1905 
(26),  88)  states  that  egg-albumen  fed  in  large  amounts  appears  in  transudates 
and  not  in  exudates,  and  can  be  detected  by  the  biological  precipitin  test.  Sugar 
is  found  more  often  in  transudates  (Sittig). 

"  Virchow's  Arch.,  1905  (179),  405. 

23 


354 


EDEMA 


excessive  fluid  escaping  in  these  conditions  carries  with  it  no  addi- 
tional proteins,  and  to  this  extent  transudates  in  statu  nascendi  are 
protein-free. 

Transudates,  even  when  produced  by  the  same  cause,  vary  in  com- 
position in  different  parts  of  the  bodj',  presumably  because  of  varia- 
tions in  the  permeability  of  the  vessels  in  different  vascular  areas;  just 
as  pleural,  pericardial,  peritoneal,  and  meningeal  fluids  normally 
differ  from  one  another.  Thus  C.  S.  Schmidt^"  found  the  composition 
of  the  transudates  in  different  pares  of  the  bodj^  of  a  patient  who  died 
of  nephritis  to  have  the  following  composition: 

Table  III 


Pleural 


Peritoneal        Subarachnoid  i  Subcutaneous 


Water 

Solids. 

Organic  matter. . 
Inorganic  matter 


963.95 

36.05 

28.50 

7.55 


978 . 91 
21.09 
11.32 

9.77 


988.70 

11.30 

3.60 

7.70 


As  in  this  case,  the  general  rule  is  that  while  che  proportion  of 
salts  remains  nearly  constant,  the  proportion  of  protein  in  edematous 
fluids  in  different  localities  varies  in  decreasing  order  as  follows: 
(1)  pleura;  (2)  peritoneum;  (3)  cerebrospinal;  (4)  subcutaneous.®^ 
In  the  last-named  location  the  specific  gravity  of  edematous  fluids 
may  be  as  low  as  1.005,  and  the  proteins  even  less  than  0.1  per  cent. 
(Hoffmann"^).  An  increase  in  solids  occurs  after  the  eft'usion  has 
existed  for  some  time,  presumably  because  of  absorption  of  water  and 
salts,  leaving  a  slowly  increasing  proportion  of  proteins.  Further- 
more, the  composition  of  the  patient's  blood  has  considerable  influ- 
ence on  the  composition  of  the  effusion;  this  is  particularly  true 
in  the  case  of  ascites  from  portal  obstruction,  the  contents  of  the  blood 
coming  from  the  intestine  during  digesoion  modifying  the  composition 
of  the  ascitic  fluid, *^^  Thus  Miiller,®*  in  a  case  of  portal  vein  throm- 
bosis, found  in  the  ascitic  fluid  of  a  patient  on  an  ordinary  mixed 
diet,  0.179  per  cent,  nitrogen;  on  a  protein-rich  diet,  0.2494  per  cent. 
N;  on  a  protein-poor  diet,  0.1764  per  cent.  N.  In  cachectic  conditions 
the  proportion  of  proteins  is  less  than  in  stronger  individuals,  and, 
as  in  the  blood  plasma,  the  albumin  decreases  more  rapidly  than  the 
globuhn  as  the  cachexia  advances  (Umber). *^^ 

Physical  Chemistry  of  Edema  Fluids. ^ — The  differences  be- 
tween transudates  and  exudates  depend  almost  solely  on  their  protein 
contents,   for  the   non-protein   elements   are    almost   identical   with 

*"  Hoppe-Seyler's  Physiol.  Chemie. 

"1  Javal  (Jour.  phys.  et  path.,  1911  (13),  50S)  places  the  fluids  in  this  order: 
serum,  peritoneal,  pleural,  sulK-utaiieous,  cercl)rospinal. 
«2  Deut.  Arch.  kliu.  Med.,  1S,S9  (44),  313. 
«=•  .See  Deni.s  and  Miiiot,  Arcli.  Int.  Med.,  1C17  (20),  879. 
«^  Deut.  Arch.  klin.  Med.,  1903  (76),  563. 
«6  Zeit.  klin.  Med.,  1903  (48),  364. 


PHYSICAL  CHEMISTRY  OF  EFFUSIONS 


355 


the  lymph  and  blood-serum,  which  naturally  must  be  so  since 
any  original  or  temporary  deviation  in  osmotic  pressure  must  be 
rapidly  cquahzed  by  diffusion.  Thus  Bodon^^  finds  the  concentra- 
tion of  the  electrolytes  nearly  constant  in  spite  of  considerable  dif- 
ferences in  composition  of  various  edema  fluids,  indicating  that  the 
serosa  permits  passage  of  inorganic  salts  ahvaj^s  in  the  same  con- 
centration, while  holding  back  the  organic  substances.  Transudates 
contain  an  excess  of  NaCl  over  other  electrolytes,  while  in  exudates 
the  proportion  of  electrolytes  other  than  chlorides  is  increased  over 
the  findings  in  transudates."  The  surface  tension  of  exudates  is 
lower  than  that  of  transudates,"^  depending  chiefly  upon  the  globulin 
content.  Rzentkowski"^  found  some  slight  differences  in  molecular 
concentration  as  indicated  bj^  the  freezing-point;  in  tuberculous  pleu- 
risy the  average  lowering  was  0.523°,  that  of  the  serum  being  —0.56"; 
in  cardiac  dropsy  the  subcutaneous  fluid  gave  —0.548°,  and  in  renal 
dropsy  —0.583°;  tuberculous  peritonitis,  —0.523°;  cirrhosis  —0.536°; 
carcinomatous  edema  —0.547°.  Of  these  figures,  the  most  significant 
is  the  comparatively  high  molecular  concentration  of  the  fluid  in 
nephritis,  supporting  the  contention  that  the  cause  of  renal  edema  is 
retention  of  crystalloids.^''  Tieken^^  has  found  the  results  in  transu- 
dates, exudates,  and  other  body  fluids  show^n  in  Table  IV, 

Table  IV 


Nature  of  fluid 


Sp.  gr. 


Freezing- 
point  of 
effusion, 
-°C. 


Freezing- 
point  of 
blood, 
-°C. 


Disease 


Pleuritic  effusion j  1,016 

Pleuritic  effusion '  1,018 

Pleuritic  effusion 1,018 

Pleuritic  effusion 1,020 

Pleuritic  effusion 1,016 

Pleuritic  effusion 1,018 

Pleuritic  effusion 1,030 

Pericardial  eft'usion 1,018 

Pericardial  eft'usion 1,016 

Pericardial  effusion 1,012 

Ascitic  fluid 1,024 

Ascitic  fluid 1,020 

Ascitic  fluid 1,018 

Ascitic  fluid 1,013 

Ascitic  fluid 1,035 

Hydrocele  fluid 1,016 

Cerebrospinal  fluid 1,018 

Cerebrospinal  fluid 1,016 

Cerebrospinal  fluid 1,020 


Cerebrospinal  fluid. 
Cerebrospinal  fluid. 
Cerebrospinal  fluid. 


1,014 
1,017 


-0.55 
-0.55 
-0.54 
-0.55 
-0.55 
-0.64 
-0.60 
-0.55 
-0.56 
-0.56 
-0.60 
-0.57 
-0.5S 
-0.62 
-0.65 
-0.56 
-0.62 
-0.64 
-0.64 
-0.56 
-0.56 
-0.56 


I     - 


0.56 
0.55 
0.56 
0.56 
0.56 
0.56 
0.58 
0.56 
0.56 
0.56 
0.56 
0.56 
0.56 
0.56 
0.58 
0.56 
0.58 
0.68 
0.64 
0.56 
0.56 
0.56 


Pneumonia,  lobar. 
Pneumonia,  lobar. 
Tuberculosis. 
Tuberculosis. 
Tuberculosis. 
Valvular  heart  disease. 
Empyema;  cyanosis. 
Pericarditis. 
Pericarditis. 
Hydropericardium. 
Cirrhosis  of  liver. 
Cirrhosis  of  liver. 
Tuberculous  peritonitis. 
Organic  heart  disease. 
General  peritonitis. 
Tuberculosis. 
Uremic  coma. 
Uremic  coma. 
Uremic  coma. 
Tuberculous  meningitis. 
Epidemic  meningitis 
Epidemic  meningitis. 


««  Pfluger's  Arch.,  1904  (104),  519;  also  see  Galeotti,  Lo  Sperimentale,  1901 
(55)   425. 

"  Griiner,  Biochem.  Jour.,  1907  (2),  383. 

«8Trevisan,  Zeit.  exp.  Path.,  1911  (10),  141. 

^^Loc.  dt.,^^  and  also  Berl.  klin.  Woch.,  1904  (41),  227. 

^^  Purulent  exudates  may  show  a  high  molecular  concentration  (  —  0.84°  in  one 
case),  due  to  decomposition  of  the  proteins  into  crvstalloids  (Rzentkowski). 
"  Amer.  Medicine,  1905  (10),  822. 


356  EDEMA 

The  very  high  figures  for  effusions  in  nephritis  and  cardiac  incom- 
petence indicate  the  concentration  of  crystalloids  in  these  fluids,  and 
support  the  behef  that  in  the  formation  of  both,  osmotic  pressure  is 
an  important  factor." 

Edema  fluids  are  usually  alkaline  except  when  bacterial  changes 
lead  to  acid  formation,  but  they  are  always  able  to  neutrahze  less  acid 
than  the  blood  of  the  same  individual  (Opie).  Bodon^^  found,  how- 
ever, that  while  they  contain  alkali  that  can  be  neutralized  by  titration 
against  acids,  yet  they  resemble  the  blood  in  being  neutral  as  far  as 
the  presence  of  free  OH  ions  is  concerned. 

Protein  Contents. — As  indicated  in  the  tables  given  previously, 
these  vary  greatly  in  quantity  in  various  fluids  ;^^  the  quantitative 
relations  of  the  different  varieties  of  proteins  have  been  less  studied. 
Serum-albumins  and  globuhns  constitute  by  far  the  largest  part  of 
the  proteins,  fibrinogen  being  scanty  except  in  some  inflammatory 
exudates,  so  that  coagulation  very  seldom  occurs  spontaneously; 
pneumococcus  exudates  seem  particularly  rich  in  fibrinogen,  which 
coagulates  rapidly  and  firmly.  The  differences  in  the  proportion  of 
different  serum  proteins  in  transudates  is  attributed  by  A.  Oswald^^ 
to  the  relative  viscosity  of  these  proteins  which  determines  their 
ability  to  pass  through  the  capillary  walls.  The  viscosity  of  serum 
proteins  varies  in  the  following  increasing  order:  albumin,  pseudo- 
globuhn,  euglobulin  and  fibrinogen;  hence  in  transudates  we  may 
find  only  the  first  two,  or  perhaps  only  the  albumin,  while  in  exudates 
the  two  latter  appear.  Joachim''^  found  in  pleural  transudates  and 
exudates  that  the  proportion  of  albumin,  euglobulin,  and  pseudo- 
globulin  is  always  lower  in  hydrothorax  than  in  pleurisy.  Of  dif- 
ferent forms  of  ascites,  the  largest  proportion  of  globuhn  and  the 
smallest  of  albumin  occur  in  cirrhosis;  while  with  carcinoma  the  pro- 
portions are  reversed.  In  general  the  albumin  is  more  abundant  than 
the  globulin, ^^  but,  as  Umber*^^  has  found,  the  proportion  of  albumin 
sinks  more  rapidly  in  cachexia  than  does  the  globulin,  corresponding 
to  the  similar  changes  in  the  blood  proteins.  The  amount  of  protein 
lost  in  exudates  is  strikingly  shown  by  one  of  Umber's  cases  of  can- 
cerous ascites;  during  one  year  the  fluid  removed  by  paracentesis 
contained  not  less  than  three  kilos  of  pure  protein,  the  patient  weigh- 
ing but  55.5  kilos. 

Several  authors  have  found  in  inflammatory  ascitic  exudates  a 
protein  having  physical  and  chemical  properties    much  resembhng 

"  Meyer  and  His  (Deut.  Arch.  klin.  Med.,  1905  (85),  149)  claim  that  the  knv- 
ering  of  the  freezing-point  is  less  than  that  of  the  blood  in  exudates  while  form- 
ing, the  same  as  the  blood  while  stationary,  and  greater  during  absorption,  which 
they  consider  indicates  a  "vital  process"  on  the  part  of  the  cells. 

"See  also  v.  Jaksch,  Zeit.  klin.  Med.,  1893  (23),  225;  Kzentkowski  (loc.  cit.)  " 

■>*  Zeit.  exp   Path.,  1910  (8),  22G. 

■'^  Pfiiiger's  Arch.,  1903  (93),  558. 

"i  See  Epstein,  Jour.  Exp.  Med.,  1914  (20),  334. 


COMPOSITION  OF  EFFUSIONS  357 

mucin;  it  has  been  especially  studied  by  Unibcr,"  who  finds  it  (juite 
similar  to  the  synovial  mucin  isolated  in  arthritis  by  Salkowski,  and 
calls  it  serosaviucin. 

Non-Protein  Organic  Contents. — Proteoses,"'  leucine,  and  tyrosine  may  be 
present  in  small  quantities  in  exudates,  being  produced  by  autolysis"  (Umber); 
and  also  mucoid  substances  (Hammarsten).  Nucleoproteins  may  be  present  from 
leucocytic  disintegration  in  exudates,  as  well  as  the  products  of  their  further 
splitting,  such  as  purines  and  phosphates,  daldi  and  Appiani'*"  found  uric  acid 
constantly  in  amounts  between  0.0055  g.  and  0.0714  g.,  in  all  exudates,  of  which 
seven  were  tuberculous  and  two  neoplastic.  In  three  transudates  amounts  from 
O.OOt)  to  0.011  g.  were  found.  Allantoin  is  said  to  have  been  found  in  exudates 
(Moscatelli),*'  but  this  is  doubtful. 

All  the  other  innumerable  components  of  plasma  may  be  found  in  edematous 
fluids;  thus  sugar^^  and  urea  (Carriere)**^  are  often  present,  as  well  as  other  ex- 
tractives. The  amount  of  urea  varies  quite  as  it  does  in  the  blood  of  the  same 
individual, ^^  and  it  seems  probable  that  all  the  crystalloid  substances  present 
in  the  blood  pass  freely  into  and  from  inflammatory  exudates,  so  that  an  equi- 
librium between  blood  and  exudates  is  approximated.*''  Sugar  is  said  sometimes 
to  be  greater  in  amount  in  transudates  than  in  the  blood,  but  in  exudates  it  is 
usually,  if  not  alwaj's,  lower  than  0.1  per  cent.*^  Glycogen  is  not  present  (Car- 
riere;.*^  By  using  more  accurate  methods  than  have  been  employed  by  most  of 
the  observers  quoted  above,  Denis  and  Minot""  found  urea,  uric  acid  and  creatinin 
to  occur  in  exudates  and  transudates  in  the  same  concentrations  as  in  the  blood, 
but  the  sugar  content  of  ascitic  fluids  is  somewhat  higher  than  that  of  the  blood. 
Creatin,  fats  and  cholesterol  are  much  lower  in  transudates  than  in  exudates  in 
which  they  approach  the  concentration  in  the  blood.  In  ascitic  fluid  the  urea, 
uric  acid  and  cholesterol  are  influenced  by  the  diet. 

Lipins. — Lecithin  is  always  present,  partly  bound  to  globulin  and  partly  free 
(Christen). ^^  Cholesterol  is  present  particularly  in  fluids  that  have  been  standing 
for  a  long  time  in  the  body,  appearing  often  as  visible  crystals  shining  in  the  fluid; 
it  probably  originates  from  degenerating  cells.  Ruppert  has  described  a  case  of 
pleural  effusion  with  1.129  per  cent,  of  cholesterol  when  tapped  the  first  time,  0.22 
per  cent,  the  second  and  0.05  per  cent,  the  third.  Hedstrom  reported  finding  in 
an  old  pleural  effusion,  4.5  per  cent,  of  chole.sterol;  one  year  later  there  was  but 
0.09  per  cent.  Zunz*'  has  described  a  carefully  studied  case  in  which  14  aspira- 
tions were  made;  the  cholesterol  content  was  about  3  per  cent,  at  first,  but  fell 
suddenly  to  0.48  per  cent,  and  then  remained  between  0.5  per  cent,  and  0.28  per 
cent.  Lecithin  varied  from  0.1  to  0.04  per  cent.  As  there  did  not  seem  to  be 
enough  cells  present  in  the  fluid  to  have  yielded  the  obtained  cholesterol  through 
their  disintegration,  Zunz  suggests  that  it  may  have  been  secreted  by  the  walls 

"' Zeit.  klin.  Med.,  1903  (48),  364;  also  Hoist,  Upsalalakar.  Forhand.,  1904, 
p.  304. 

'8  Opie,  Jour.  Exp.  Med.,  1907  (9),  391. 

'3  Histidine  and  arginine  were  found  in  a  carcinomatous  exudate  by  Wiener 
(Biochem.  Zeit.,  1912  (41),  149). 

8°  Riforma  Med.,  1904,  p.  1373;  also  Carriere,  Compt.  Rend.  Soc.  Biol.,  1S99 
(51),  467. 

81  Zeit.  physiol.  Chem.,  1899  (13),  202. 

82  Sugar  was  found  in  only  8  of  23  fluids  by  Sittig  (Biochem.  Zeit.,  1909  (21), 
14) ;  but  is  present  in  pulmonary  edema  fluid  in  proportion  equal  to  or  even  greater 
than  the  blood  (Ivleiner  and  Meltzer). 

83  Javal  and  Adler,  Compt.  Rend.  Soc.  Biol.,  1906  (61),  235;  Rosenberg,  Berl. 
klin.  Woch.,  1916  (53),  1314. 

8^  Wells  and  Hedenburg,  Jour.  Infect.  Dis.,  1912  (11),  349;  Scheel,  Nord.  Med. 
Laeg.,  1916  (77),  610. 

85  Hegler  and  Schumm,  Med.  Klinik,  1913  (9),  1810. 

86  Compt.  Rend.  Soc.  Biol.,  1899  (51),  467. 

87  Arch.  Int.  Med..  1917  (20),  879. 

88  Cent.  f.  inn.  Med.,  1905  (26),  329. 

83  Travaux  Ambulance  de  L'Ocean,  La  Panne,  1918,  Tome  II,  Fasc.  1. 


358  EDEMA 

of  the  cavity.  Weems^°  has  described  a  similar  case,  with  1.39  per  cent,  in  the 
first  fluid  drawn,  but  smaller  amounts  in  fluids  withdrawn  later;  this  patient  had 
a  marked  hypercholesterolemia.  ArnelP^  found  0.41  i)er  cent,  of  cholesterol  in  a 
tuberculous  pleurisy.  In  most  of  these  cases  some  fats  have  been  present,  Weems 
finding  0.33  per  cent,  and  Ruppert  0.36  per  cent. 

Toxicity. — Contrary  to  earlier  ideas,  transudates  are  not  demonstrably  toxic, 
even  in  nephritis  (Baylac,^^  Boy-Teissier,.'^  Lafforcade'^),  and  therefore  the  toxic 
manifestations  frequently  observed  after  reduction  of  edema  in  nephritis,  and 
ascribed  to  absorption  of  poisons  in  the  transudates,  are  probably  due  to  some 
other  cause.  In  inflammatory  exudates,  of  course,  the  causative  agents  as  well 
as  the  products  of  cell  destruction  render  the  fluids  poisonous. 

Enzymes  and  Immune  Bodies. — All  the  enzymes  of  the  plasma  may  appear 
in  edematous  fluids,  being  in  all  cases  probably  more  abundant  in  exudates  than 
in  transudates.  According  to  Carriere,^^  oxidases  are  inconstant,  even  in  exu- 
dates. Lipase  is  said  to  be  much  more  abundant  in  exudates  than  in  transudates.  ^^ 
(Concerning  proteolytic  enzymes  see  "Autolysis  of  Exudates,"  Chap,  iii.)  The 
various  immune  bodies,  cytotoxins,  hemolysins,  bacteriolj'sins,  agglutinins,  etc., 
seem  to  pass  freely  into  both  transudates  and  exudates,  and  their  presence  is  not 
characteristic  of  either, ^^  but  as  a  rule  the  proportion  is  much  higher  in  exudates.^* 
Peptid-splitting  enzymes  are  usually  found  in  such  fluids, ^^  especially  tuberculous 
exudates,'  and  these  enzymes  seem  to  be  different  from  both  erepsin  and  trypsin. 
Probably  this  type  of  enzyme  is  more  often  present  than  trypsin.  Antitryptic 
activity  is  usually  high,  unless  exhausted  by  the  presence  of  much  trypsin  from 
cell-rich  exudates.  Purulent  fluids  are  usually  poor  in  opsonins;-  in  non-purulent 
fluids  the  opsonin  content  varies  with  the  amount  of  proteins.'  Turpentine  exu- 
dates may  sometimes  be  more  strongly  bactericidal  than  the  serum  of  the  same 
animal.''  Exudates  usually  contain  about  as  much  complement  as  the  serum, 
but  in  suppuration  the  complement  disappears;  transudates  contain  little  of  either 
complement  or  hemolysins.^ 

Precipitin  Reactions,  etc. — Edematous  fluids  have  been  often  used  as  a  source 
of  material  in  immunizing  animals  against  human  proteins.  The  precipitins  thus 
formed  are  specific  for  human  serum  or  for  the  proteins  of  the  effusion,  but  can 
not  be  used  to  differentiate  a  transudate  from  an  exudate,  or  a  hj'drothorax  fluid 
from  an  ascites  fluid  (Quadrone).^  Immune  bodies,  complement,  agglutinins  and 
antitoxins  are  present  in  effusions;  e.  g.,  the  common  use  of  blister  fluid  for  the 
Widal  test.  Furthermore,  according  to  Hamburger,^  edema  fluid  is  distinctly 
more  bactericidal  than  normal  lymph. 

Varieties  of  Edematous  Fluids^ 

On  the  preceding  pages  have  been  mentioned  the  chief  differences 

in  the  characters  of  the  effusions  in  the  usual  sites,  ^  with  their  varia- 

90  Amer.  Jour.  Med.  Sci.,  1918  (156),  20. 
"  Hygiea,  1917  (79),  737. 
'••2  Compt.  Rend.  Soc.  Biol.,  1901  (53),  519. 
'■>^  Ibid.,  1904  (56),  1119. 
9^  Gaz.  heb.  Med.  et  Chir.,  Jan.  28,  1900. 
"^  Compt.  Rend.  Soc.  Biol,  1899  (51),  561. 

9"  Zeri,  II  Policlinico,  1903  (10),  No.  11;  Memmi,  Clin.  med.  Ital.,  1905,  Xo.  3; 
Galletta,  Chn.  med.  Ital.    1911  (50),  143. 

'■"  Granstrom,  Inaug.  Dissert.,  St.  Petersburg,  1905. 

98  Not  corroborated  by  Ludke,  Cent.  f.  Bakt.,  1907  (44),  268.  See  also  Delrez 
Bull.  acad.  Rov.  Med.  Belg.,  1919  (29),  733. 

99  Hall  and  WiUiamson,  Jour.  Path,  and  Bact.,  1911  (15),  351. 
iSee  H.  Koch,  Zcit.  Kinderhoilk.,  1914  (10),  1. 

2  Opie,  Jour.  Expcr.  Med..  1907  (9),  515. 

3  Bohmc,  Dcut.  Arch.  klin'.  McmI.,  1909  (96),  195. 
Mlastaedt,  Zcit.  Inimunitat.,  1912  (13),  421. 

"■  Aronstamm,  Cent.  f.  Bakt.,  1914  (74),  326. 
eCent.  f.  Bakt.  (Ref.),  1905  (3()),  270. 
'  Virchovv's  Arch.,  1899  (156),  32S). 

"  Chemistry  of  Pus  and  Sputum  are  discussed  under  Inflammation,  Chapter  xi. 
9  Literature  and  r6sume  on  pleuritic  exudates,  see  Ott,  Chcm.   Pathol,  dor 
Tubcrc,  1903,  {).  392. 


COMPOSITION  OF  EFFUSIONS  359 

tions  in  protein  contents,  which  variation  agrees  with  StarUng's  state- 
ment that  the  permeabiHty  of  the  capillary  wall  for  proteins  differs 
normally  in  different  localities.  Some  of  the  other  effusion  fluids  not 
mentioned    previously  have  particular  properties  of  some  interest. 

Subcutaneous  Efifusions.^" — When  of  non-inflammatory  origin 
these  are  vcrj' watery,  having  ordinarily  a  protein  content  of  from  0.1 
to  0.2  gm.  per  100  c.c,  there  being  more  globulin  in  nephritic  than  in 
cardiac  dropsy.  The  non-coagulable  nitrogen  and  chloride  content 
are  not  so  high  as  in  the  blood  of  the  same  patients,  but  the  ash  is  the 
same  as  that  of  the  serum.  The  specific  gravity  may  be  as  low  as 
l.OOo,  but  the  solids  increase  with  the  duration  of  the  edema. 

Hydrocele  and  Spermatocele  Fluids. — These  have  been  studied 
particularly  by  Hammarsten,  who  found  the  average  result  of  analyses 
of  seventeen  hj'drocele  fluids  and  four  spermatocele  fluids  as  follows: 

Table  V 

Hydrocele  Spermatocele 

Water... 938.85                986.83 

Solids 61.15                   13.17 

Fibrin 0.59 

Globulin 13.25                    0.59 

Seralbumin 35.94                     1.82 

Ether-extractive  bodies 4 .  02  ] 

Soluble  salts 8.60                  10.76 

Insoluble  salts 0 .  66  J 

Marchetti"  found  in  ten  specimens  of  hydrocele  fluid  rather  higher  results  for 
the  solids  than  did  Hammarsten.  He  found  57.8  to  104.2  p.  m.  of  solids,  contain- 
ing organic  sub.stances  48.8  to  95.02,  and  inorganic  substances  8. 10  to  9.56;  proteins, 
33.5  to  90.19;  ratio  of  globulin  to  albumin  as  2.56  to  9.11.  Among  the  pro- 
teins is  found  1  to  4  p.  m.  that  is  not  precipitated  by  heat.  Corresponding  with 
the  analytic  results,  the  specific  gravity  of  hydrocele  fluid  is  higher,  1.016  to  1.026 
as  against  1.006  to  1.010  for  spermatocele  fluid.  Cholesterol  is  often  abundant  in 
hydrocele  fluids,  appearing  to  the  naked  eye  as  glistening  scales.  Patein'^  found 
sugar  in  most  specimens  of  hydrocele.  Apparently  hydrocele  fluid  stands  inter- 
mediate in  properties  between  transudates  and  exudates.'^  Usually  it  contains 
but  little  of  the  immune  bodies  from  the  blood  (Delrez).^* 

Meningeal  Effusions. ^^ — Normal  meningeal  fluid  differs  from  all 
other  serous  fluids  in  being  clear  and  watery,  in  its  low  specific  gravity 
(1.004  to  1.007),  in  containing  but  a  trace  of  protein  which  is  chiefly 
globulin  (with  a  trace  of  proteose  (?)  ),  and  0.05-0.13  per  cent,  of  a 
reducing  substance  that  is  probably  glucose, ^^  which  is  decreased  in 

1°  See  Epstein,  Jour.  Exper.  Med.,  1914  (20),  334. 

11  Lo  Sperimentale,  1902  (56),  297. 

12  Jour,  pharm.  et  chim.,  1906  (23),  239;  also  Compt.  Rend.  Soc.  Biol.,  1906 
(60),  303. 

13  Vecchi,  Gaz.  Med.  Ital.,  1912  (63),  211;  Epstein,  Jour.  Exp.  Med.,  1914  (20), 
344. 

1^  Resume  by  Blumenthal,  Ergeb.  der  Physiol.,  1902  (1),  285;  Blatters  and 
Lederer,  Jour.  Amer.  Med.  Assoc,  1913  (60),  811;  Herrick  and  Dannenberg,  ibid., 
1919  (73),  1321;  Levinson,  Amer.  Jour.  Dis.  ChUd.,  1919  (18),  568;  Becht,  Amer. 
Jour.  Physiol.,  1920  (51),  1. 

1*  Schloss  and  Schroeder,  Amer.  Jour.  Dis.  Child.,  1916  (11),  1;  Hopkins, 
Amer.  Jour.  Med.  Sci.,  1915  (150),  847. 


360  EDEMA 

acute  suppurative  meningeal  inflammation  (Jacob). ^^  There  is  nor- 
mally in  the  adult  from  60  to  150  cc,  and  Frazier  estimates  that  from 
360  to  720  cc.  is  secreted  daily.  HalHburton  gives  the  following  analyses 
of  pathological  accumulations  of  such  fluids : 

Table  VI  (Spina  bifida) 

Case  1  Case  2  Case  3 

Water 989.75  989.877  991.658 

Solids 10.25  10.123  8.342 

Proteias 0.842  1.602  0.199 

Salts               1  Q  .^^  /  0.631  3.C28 

Extractives/ ^■^'^^  \7.89f  5.115 

The  percentage  of  solids  in  spina  bifida  is  thus  a  little  higher  than 
in  normal  meningeal  fluids.  In  hydrocephalus  the  percentage  of  solids 
is  rather  greater,  as  seen  in  Table  VII. 

Table  VII  (Hydrocephalus; 

Case  1  Case  2  Case  3 

Water 986.78  984.59  980.77 

Solids 13.22  15.41  19.23 

Proteins  and  extractives 3.74  6.49  11.35 

Salts 9.48  8.92  7.88 

Normal  cei'ebrospinal  fluid  seems  to  be  h3'pertonic  to  the  serum  of 
the  same  animaP^  and  slightly  more  alkaline  than  the  blood.  ^^  In 
meningitis  the  alkalinity  is  often  lowered. ^^  The  alkaU  reserve  is 
nearly  constant  in  systemic  diseases,  except  diabetes  (McClendon),'° 
and  is  practically  the  same  as  that  of  the  blood.  By  gas  chain  meas- 
urements Levinson-i  found  the  spinal  fluid  almost  neutral  (pH  = 
7.4-7.6);  in  epidemic  meningitis  it  is  7.3-7.4.  According  to  Fuchs 
and  Rosenthal--  the  average  freezing-point  of  the  cerebrospinal  fluid 
is  lowered  about  the  same  in  all  diseases  (A  =  —0.52°  to  0.54°)  ex- 
cept in  tuberculous  meningitis,  where  it  is  much  less  (average  —0.43°). 
The  amount  of  potassium  is  about  the  same  as  in  the  blood, -^  and  not 
increased  in  degenerative  diseases  of  the  central  nervous  system;-* 
after  death  the  amount  is  much  increased  by  post-mortem  changes. 
Calcium  is  almost  constant  at  5  mg.  per  100  cc,  or  about  one-half 
as  much  as  in  the  plasma."  In  diseases  associated  with  destruction 
of  brain  tissue,  such  as  general  paralysis  and  epilepsy,-^  choline  or  some 

'^  Brit.  Med.  Jour.    1912  Oct.  26. 

»'  Ravaut,  Presse  rnod.,  1900  (8;,  128;  Zanier,  Cent.  f.  Fhysiol.,  1896  (10),  353. 
•8  Hurwitz  and  Tranter,  Arch.  Int.  Med.,  1916  (17),  828. 
>»  Levinson,  Arch.  Pediatrics,  1916  (33),  241. 
2"  Jour.  Amer.  Med.  Assoc,  1918  (70),  977. 
2>  .jour.  Infect.  Dis.,  1917  (21),  556. 
"  Wion.  mcd.  Presse,  1904  ^45),  2081  and  2135. 
"Myers,  Jour.  Biol.  Cheni.,  1909  (6),  115,  literature. 
"^  Rosenbloom  and  Andrews,  Arch.  Int.  Med.,  1914  (14;,  536. 
"Halverson  and  Borfreini,  Jour.  Biol.  Chem.,  1917  (29),  337. 
2*  Concerning    si)inai  fluid  in  cpilcp.sy  see  Larkin  and  Cornwall  (Jour.  Lab. 
Clin.  Med.,  1919  (4),  352. 


MENINGEAL  EFFUSIONS  361 

other  base"  may  be  found  in  the  spinal  fluid.  (See  "Choline, "  Chap. 
iv.)  ^ 

Under  pathological  conditions  the  amount  of  protein  varies  greatly 
and  to  some  extent  characteristically.  Thus,  in  syphilis  the  euglobulin 
is  so  greatly  increased  that  it  is  readily  identified  by  various  precip- 
tation  methods,-^  while  in  more  acute  inflammations  fibrinogen  ap- 
pears.-* According  to  Mott^''  the  fluid  is  especially  rich  in  nuclein 
in  progressive  paral^'sis,  and  lipoids  are  increased  in  the  fluid  in  do- 
generations  of  the  central  nervous  system.  Pathological  fluids  show 
also  specific  alterations  in  their  colloidal  property  of  preventing  pre- 
cipitation of  colloidal  suspensions  b}'  electrolytes  (the  "Goldzahl" 
of  Zsigmondy).'^  The  surface  tension  is  higher  than  that  of  the 
serum  and  is  not  characteristically  altered  in  disease. ''^  The  increased 
organic  matter  of  pathological  fluids  raises  the  permanganate  reduction 
index."  In  epidemic  meningitis  there  is  more  positively  charged 
protein  while  in  tuberculous  meningitis  there  is  more  negatively 
charged  protein,  which  can  be  distinguished  by  suitable  precipitants 
(Tashiro  and  Levinson).^^ 

Cholesterol  can  be  found  in  all  cases  of  mental  disease,  the  amount 
not  bearing  any  relation  to  the  type  of  ps3'chosis  (Weston)  ;^'  ordinar- 
ily' 0.2  to  0.7  mg.  per  100  c.c.  is  found.  The  changes  in  PoOs  content 
in  disease  are  doubtful,^^  while  the  amount  of  reducing  substances  is 
said  to  be  increased  in  disease. ^'^  In  general  the  inflammatory  fluids 
in  the  spinal  canal  resemble  exudates  elsewhere,  but  usually  the  con- 
centration of  the  different  components  is  relatively  low,  except  the 
chlorides. ^^  Normal  cerebrospinal  fluid  contains  no  antiprotease  (for 
leucoprotease),  as  does  the  fluid  in  many  cases  of  chronic  inflamma- 
tions; in  acute  inflammation  proteases  ma}-  appear  (Dochez^*).  Pep- 
tid-splitting  enzymes  are  especially  abundant  in  meningitis.  ■*"  Anti- 
bodies pass  from  the  serum  into  the  cerebrospinal  fluid  only  in  minimal 
amounts  or  not  at  all,  except  when  inflammatory  exudation  occurs, 
and  even  then  the  antibody  concentration  is  usually  low,'*^  and  even 

2"  Kaufmann,  Zeit.  physiol.  Chem.,  1910  (66),  3-13;  Laignel-Lavastine  and 
Lasusse,  Compt.  Rend.  Soc.  Biol,  1910  (68),  803. 

28  See  Xoguchi,  Jour.  Exp.  Med.,  1909  (11),  604. 

='  See  Mestrezat,  Rev.  d.  Med.,  1910,  p.  189;  Kaflfka,  Deut.  med.  Woch.,  1913 
(39),  1874. 

3°  Lancet.  July  9,  1910. 

"  Lange,  Zeit.  Chemother.,  1912  (1),  44;  Spat,  Zeit.  Immunitat.,  1915  (23),'426; 
Vogel,  Arch.  Int.  Med.,  1918  (22),  496. 

32  Ivisch  and  Remertz,  Miinch.  med.  Woch.,  1914  (20),  1097. 

"  See  Hoffman  and  Schwartz,  Arch.  Int.  Med.,  1916  (17),  293. 

3*  Jour.  Infect.  Di.s.,  1917  (21),  571. 

35  Jour.  Med.  Res.,  1915  (33),  119. 

3®  Apelt  and  Schumm,  Arch.  Psj-chiat.  u.  Xervenkr.,  ISOS  (44),  845. 

"  Jacob,  Brit.  Med.  Jour.,  Oct.  26,  1912. 

3s  Javal,  Jour.  phv.s.  et  path,  gen.,  1911  (15),  508. 

39  Jour.  Exp.  Med.,  1909  (11),  718. 

">  Major  and  Xobel,  Arch.  Int.  Med.,  1914  (14),  383. 

*iLemaire  and  Debre,  Jour,  physiol.  et  path,  g^n.,  1911  (13),  233. 


362  EDEMA 

simple  chemicals  enter  the  normal  spinal  fluid  but  very  httle,^-  ex- 
cept perhaps  alcohol. ^''^  According  to  Rosenbloom'*'*  there  is  no  crea- 
tin  or  creatinine.  It  contains  normally  from  2  to  4  mg.  of  amino-N 
per  100  c.c,  or  about  half  that  in  the  blood,  without  definite  changes 
in  syphilis. ^^  There  is  almost  the  same  amount  of  urea  as  in  the 
serum  of  the  same  person,  i.  e.,  20"  to  42  mg.  per  100  c.c.*®  In  uremia 
the  non-protein  constituents  of  the  spinal  fluid  increase  with  those  of 
the  blood,  but  to  a  less  degree.  Substances  giving  the  ninhydrin 
test  appear  in  meningitis,'*^  but  Rosenberg  states  that  even  with  the 
highest  indicanemia"*^  no  indican  is  found  in  the  spinal  fluid.  Sugar 
is  present  in  from  0.07  to  0.085  per  cent,  and  is  not  modified  signifi- 
cantly in  mental  diseases;*^  it  is  reduced  in  meningitis  but  increased  in 
uremia.^"  There  is  only  a  very  small  amount  of  diastase,  not  bearing 
any  constant  relation  to  the  cell  count. '^^ 

Xanthochromia. — In  cases  of  retention  of  spinal  fluid,  usually  in  a 
lumbar  cul-de-sac,  it  may  assume  a  yellow  color  although  free  from 
blood  pigment,  containing  much  globulin  and  coagulating  spontane- 
ously (Froin's  syndrome) .  The  color  is  apparently  due  to  concentra- 
tion of  plasma  held  for  some  time  in  the  spinal  canal  and  may  be 
from  bilirubin.  ^^  Most  usually  this  condition  accompanies  tumor  of 
the  spinal  cord.^^ 

Wound  secretions  obtained  from  large  aseptic  wounds,  mostly  amputation 
stumps,  have  been  studied  by  Lieblein.''''  The  reaction  is  generally  alkaline, 
globulin  and  albumin  abundant,  but  fibrinogen  scanty,  total  nitrogen  being  less 
than  that  of  the  blood  and  decreasing  from  day  to  day;  the  proportion  of  albumin 
increases  and  globulin  decreases  as  healing  progresses.  Occasionally  albumoses 
were  found,  but  only  on  the  first  day  in  aseptic  wounds;  if  found  later,  they  gener- 
ally were  antecedent  to  suppuration  (concerning  suppuration  see  "Inflammation,  " 
Chap.  xi). 

Blister  fluid  is  generally  rich  in  solids  and  proteins  (40-65  p.  m.).  In  a  burn 
blister  Morner^^  found  50.31  p.m.  proteins,  among  which  were  11.59  p.  ni.  globulin 
and  but  0.11  p.  m.  fibrin;  also  a  substance  reducing  copper  oxide,  but  not  pyro- 
catechin.  By  refractometric  determinations  the  amount  of  protein  in  blister 
fluids  is  in  direct  proportion  to  the  amount  in  the  blood. ^''     Antibodies  of  all 

"  See  Rotky,  Zeit.  klin.  Med.,  1912  (75),  494. 

"  Schottmiillerand  Schumm,  Neurol.  Zbl.,  1912  (31),  1020. 

"  Biochem.  Bull.,  1916  (5),  22. 

«  Ellis,  elal,  .Jour.  Amer.  Med.  Assoc,  1915  (64),  126. 

«  Ellis  and  Cullen,  Jour.  Biol.  Chem.,  1915  (20)  511. 

^^  Nobel,  Miinch.  med.  Woch.,  1915  (62),  1355,  1786. 

^»  Berl.  kl.  Woch.,  1916  (53),  1314. 

^'  Weston  Jour.  Med.  Res.,  1916  (35),  199;  Kraus  and  Corneille,  Jour.  Lab. 
Clin.  Med.,  1916  (1),  685. 

"  Leopold  and  Bernhard,  Amer.  Jour.  Dis.  Chil.,  1917  (13),  34.  Discussion  of 
chemistry  of  spinal  fluid  in  children. 

"  Jicsclike  and  Pincussohn,  Deut.  med.  Wochs.,  1917  (43),  8;  Katakura,  Kyoto 
Jour.  Med.  Sci.,  1916  (13),  1. 

"  Bauer  and  Spiegel,  Deut.  Arch.  klin.  Med.,  1919  (129),  18. 

6»  Review  by  Sprunt  and  Walker,  Bull.  Johns  Hop.  IIosp.,  1917  (28),  80;  Elsberg 
and  Rochfort,  Jour.  Amer.  Med.  Assoc,  1917  (68),  1S02. 

"  Beit.  klin.  Chir.,  1902  (35),  43. 

"  Hammarsten,  "Physiological  Chemistry." 

59  Engel  and  Orszag,  Zeit.  klin.  Med.,  1909  (67),  175. 


CHYLOUS  EFFUSIONS  363 

sorts  seem  to  pass  readily  into  blister  fluids''  although  the  complement-fixation 
reaction  is  not  so  stroiiij  as  with  the  blood."* 

Hydrops  of  Gall  Bladder. — The  watery  fltiid  contains  99  per  cent,  water,  a 
mucin-like  substance,  but  no  otiier  proteins  and  no  bile  acids." 

Fetal  Bronchiectasis. — The  fluid  resembles  closely  liquor  amnii.'" 

Chylous  Effusions.^' — Fat  may  be  present  in  effusions  in  sufficient 
quantity  to  cause  a  milky  appearance,  either  from  escape  of  chyle 
from  a  ruptured  or  obstructed  thoracic  duct,  or  through  fatty  degen- 
eration of  the  cells  in  the  effusion  or  the  lining  of  the  walls  of  the 
cavity.  The  former  are  designated  as  chylous,  the  others  as  chyU- 
form  or  adipose  fluids,  but  it  is  not  always  easy  to  distinguish  be- 
tween them.  The  composition  of  the  fluids  in  true  chj'lous  exudates 
will  var}'  according  to  the  food  taken  and  the  amount*  of  fat  the  food 
contains,  and  will  resemble  the  composition  of  chyle,  except  to  the 
extent  that  it  is  modified  by  the  effusion  or  absorption  going  on  in  the 
cavity.  They  are  characterized  by  strong  bactericidal  powers  as  evi- 
denced by  lack  of  putrefaction  after  long  standing. 

Analyses  of  human  chyle  are  scanty.  Panzer^^  found  90.29-94.53  per  cent, 
water;  5.47-9.71  per  cent,  solids;  0.80-1.04  per  cent,  inorganic  salts;  2.16  per  cent, 
coagulable  protein;  6.59  per  cent,  ether-soluble  material;  also  diastatic  enzyme, 
soaps,  and  occasionally  traces  of  cholesterol,  lecithin,  and  sugar.  Carlier,^^  in 
a  specimen  from  a  child,  obtained  very  similar  results,  except  that  the  salts 
were  much  less  abundant.  The  proteins  and  fats  vary  greatly  with  the  diet;  thus 
Sollmann^^  found  variations  in  the  proteins  from  1.85  to  6.5  per  cent. 

Edwards^'^  found  that  of  31  definitely  estabUshed  cases  of  chylous 
or  chyliform  ascites  studied  at  autopsy,  in  21  there  was  established  the 
existence  of  a  rupture  in  the  thoracic  duct  or  lacteals.  Boston^^  in 
1905  was  able  to  collect  126  cases,  including  both  chylous  and  chyliform 
ascites,  and  notes  an  associated  eosinophilia  in  a  case  studied  b}-  him. 
Chylous  ascites  fluid  often,  but  not  always  contains  sugar,"  but 
it  maj^  disappear  after  having  once  been  present;  the  amount  of  fat 
is  small,  usually  about  1  per  cent.,  and  the  fluid  is  rich  in  sohds. 
If  due  to  a  ruptured  thoracic  duct,  it  may  be  possible  to  detect  special 
fats  taken  in  the  food,  e.  g.,  butter-fats  (Straus).®^     The  reaction  is 

"  Eisenberg,  Deut.  med.  Woch.,  1909  (35),  613. 

58  Buschke  and  Zimmermann,  Med.  Ivlinik,  1913  (9),  1082. 

59Sjoquist,  SvenskaLak.  Handl.,  1916  (42),  1291. 

«o  Koeckert,  Amer.  .Jour.  Dis.  Chil.,  1919  (17),  95. 

"  Literature  bv  Gandin,  Ergeb.  inn.  Med.,  1913  (12),  218. 

"  Zeit.  phvsiol.  Chem.,  1900  (30),  113. 

"  British  xMed.  Jour.,  1902  (ii),  175. 

«<  -\mer.  Jour.  Phvsiol.,  1907  (17),  487;  see  also  Hamill,  Jour.  Physiol.,  1906  (35), 
151. 

"Medicine,  1895  (1),  257;  also  see  "Chem.  u.  morph.  Eigenschaften  fett- 
haltige  Exsudaten,"  St.  Mutermilch,  Warschau,  1903;  Comey  and  McKibben, 
Boston  Med.  and  Surg.  Jour.,  1903  (148),  109. 

«  Jour.  .'^ler.  Med.  Assoc,  1905  (44),  513. 

«'  For  example,  v.  Tabora  (Deut.  med.  Woch.,  1904  (30),  1595)  found  as  high 
as  0.864  per  cent,  of  sugar  in  a  typical  case. 

"  Arch.  Physiol,  et  Pathol.,  1886  (Ser.  3,  vol.  8),  367. 


364  EDEMA 

usually  alkaline  or  neutral,  and  some  specimens  coagulate  spontane- 
ously. Specific  gravity  varies  from  1.007  to  1.040,  the  average  being 
about  1.017.  Perhaps  the  most  important  characteristic  is  the  varia- 
tion produced  by  changes  in  diet.^^  Zdarek'^"  found  in  a  chyle-cyst 
2.7  per  cent,  of  fats,  7.2  per  cent,  of  proteins,  and  0.05  per  cent,  of 
sugar;  feeding  of  fats  increased  their  amount  in  the  cyst  and  star- 
vation decreased  it.  Schumm''^  found  in  the  solids  of  such  a  cyst 
35.76  per  cent,  of  fat,  some  of  which  was  in  the  form  of  calcium  soap. 

Chyloihorax  fluid  is,  of  course,  quite  similar  to  that  of  chylous  ascites.  Thus, 
Buchtala^^  found  91.34  per  cent,  of  water,  8.66  per  cent,  solid,  4.86  per  cent, 
protein,  2.5  per  cent,  fat,  0.26  per  cent,  cholesterol,  and  0.94  per  cent.  ash.  Similar 
figures  were  obtained  by  Salkowski^^  and  others. 

Chyluria,''*  which  seems  to  depend  upon  an  abnormal  communication  between 
the  lymphatics  of  the  receptaculum  chyli  and  the  kidney,^^  shows  no  particular 
chemical  features  beyond  those  of  an  admixture  of  a  considerable  amount  (100  to 
1000  c.c.  per  day)  of  chyle  with  the  urine.  Carter"'  found  the  amount  of  fat  in 
the  urine  to  rise  with  increase  of  fat  in  the  food.  Pecker^^  observed  a  rise  from  a 
former  average  of  1.5  gm.  fat  per  liter  to  9.75  gm.  after  eating  oils  and  milk.  In 
some  cases  chyle  escapes  directly  into  the  bladder  or  ureter  from  the  lymphatics, 
in  others  the  fat  may  be  excreted  directly  from  the  blood,  independent  of  lymphatic 
abnormality;  in  some  cases  the  fluid  entering  the  urine  is  true  chyle  and  in  others  it 
is  lymph. 

Ascites  adiposus  is  characterized  by  the  absence  of  sugar  and  by  a  higher 
percentage  of  fat,  the  maximum  observed  being  6.4  per  cent.  It  is  ascribed  to 
fatty  metamorphosis  of  cells,  particularly  in  carcinomatous  and  tuberculous  exu- 
dates; Edwards  was  able  to  show  experimentally  that  a  transudate  may  change 
from  serous  to  cellular,  and  later  come  to  contain  fat. 

Pseudochylous  effusions  are  also  observed,  not  only  in  the  abdominal  and 
thoracic  cavities,  but  even  in  the  fluid  of  the  edematous  legs  and  scrotum ;  these 
resemble  chylous  fluids  in  being  turbid  or  milky,  but  are  said  to  contain  little  or  no 
fat.  The  turbidity  is  ascribed  chiefly  to  lecithin,  which  is  largely  combined  with 
the  pseudoglobulin  of  the  fluid  (.Toachim).'^  Possibly  in  some  cases  the  turbidity 
is  partly  or  largely  (Poljakoff)'*  due  to  poorly  dissolved  proteins.  Strauss*" 
has  noted  the  occurrence  of  this  form  of  ascites  particularly  in  chronic  parenchy- 
matous nephritis,  but  believes  the  turbidity  has  a  local  origin.  Hammarsten  has 
observed  turbidity  due  to  mucoid  substances,  as  also  have  Gouraud  and  Corset.*' 
The  pseudo-chylous  effusions  have  a  lower  freezing  point,  a  lower  specific  gravity, 

^^  A  sample  of  the  composition  of  1  liter  of  chylous  ascitic  fluid  is  shown  by 
the  analysis  in  the  case  studied  by  Comey  and  McKibben  {loc.  cit) :  Specific 
gravity,  1.010;  solids,  21  gm.;  protein  9.75  gm.;  urea,  1.28  gm.;  fat,  1.45  gm.; 
inorganic  matter,  8  gm.;  peptone  (?)  and  sugar,  present;  fibrinogen,  mucin, 
nucleo-albumin,  and  uric  acid  absent. 

'«  Zeit.  f.  Heilk.,  1906(27),!. 

'1  Zeit.  physiol.  Chem.,  1906  (49),  266. 

^'  Zeit.  physiol.  Chem.,  1910  (67),  42. 

"  Virchow's  Arch.,  1909  (198),  189;  also  Tuley  and  Graves,  Jour.  Amer.  Med 
Assoc,  1916  (66),  1844;  Patein,  Jour,  pharm.  Chim.,  1915  (11),  265. 

''*  Review  of  literature  by  Sancs  and  Kahn,  Arch.  Int.  Med.,  1916  (17),  181. 

"  See  Magnus-Levy,  Zeit.  klin.  Med.,  1908  (66),  482. 

""^  Arch.  Int.  Med.,  1916  (18),  541. 

"Jour,  pharm.  chim.,  1917  (16),  139.     See  also  Patein   ibid.,  1917  (16),  230 

78Muncli.  mod.  Woch.,  1903  (50),  1915;  also  Christen.  Cent.  f.  inn.  Med.  1905 
(26),  329;  Wallis  and  Scholberg,  ()uart.  Jour.  Med.,  191f  (3),  301;  1911  (4),  153. 

'»  Fortschr.  d.  Med.,  1903  (21;,  1081;  also  Haushalter,  Compt.  Rend.  Soc.  Biol., 
1910  (68),  550. 

8«  Note  to  Poljakoff's  article;"  also  Biochem.  Centr.,  1903  (1),  437. 

8'  Coin))!.  Rend.  Soo.  Biol.,  1906  (60),  23. 


PNEUMOTHORAX  365 

lower  fat  and  greater  lecithin  content  than  typical  chylous  ascites.  Gandin," 
however,  questions  the  possibility  of  always  differentiating  the  three  types  of  turbid 
fluids  as  above  indicated.  Collecting  all  the  recorded  analyses  in  the  literature 
he  finds  wide  discrepancies,  as  indicated  in  the  following  table:  (The  maximum  and 
minimum  percentage  figures  are  given  for  each  component  determined  quantita- 
tively, with  the  average  in  parentheses.) 

Adipose 
Chylous  (Chyliform)  Pseudochylous 

Ether  extract  0.065-9.2(1.65)  0.1-4.3(1.15)  0.007-1.86(0.25) 

Cholesterol  +  in  7,  —  in  2  +  in  4  +  in  3,  —  in  2 

Lecithin  -j-  in  4,  —  in  1  +  in  3  +  in  20,  —  in  2 

Sugar  4-  in  46,  —  in  28  -f  in  1,  —  in  4  +  in  15,  —  in  14 

Dry  residue  3.1-10.6(6.2)  1.6-11.7(5.1)  1.2-7.6(2.9) 

Protein  0.9-7.7(3.5)  0.6-6.8(3.0)  0.1-4.2(1.4)' 

"Pepton"  +  in  6,  -in  4  +  in  1,  -  in  2  +  in  1,  -  in  5 

Ash  0.1-1.0  (0.59)  0.45-1.03  (0.65)  0.49-0.90  (0.73) 

It  is  quite  evident  that  although  the  pseudochylous  fluids  usually  contain  little 
fat,  they  often  contain  more  than  the  minimal  content  found  in  the  other  forms. 
Each  type  of  fluid  overlaps  the  others  in  one  respect  or  another.  Gandin  states 
that  to  produce  a  turbid  fluid  but  0.01-0.1  per  cent,  of  finely  emubionized  fat 
is  necessary,  and  he  believes  that  milky  fluids  always  mean  admixture  of  chyle, 
rejecting  the  terms  pseudochylous  and  chyliform  as  unwarranted.  He  admits 
that  fluids  may  contain  droplets  of  fats  not  emulsionized,  and  hence  not  milky, 
which  may  be  properly  called  adipose  fluids.  There  are  no  characteristic  chemical 
differences  in  the  fats  extracted  from  the  different  types  of  fluids. 

Chemistry  of  Pneumothorax 

In  connection  with  the  subject  of  exudates  the  above  topic  may  appropriately 
be  considered.  The  composition  of  the  gases  found  in  the  pleural  cavity  in  pneu- 
mothorax will  necessarily  vary  greatly  according  to  the  cause.  If  the  pleural 
cavity  is  in  free  communication  with  the  exterior,  the  gas  will  be  simply  slightly 
modified  air;  for  example,  Ewald^^  found  the  following  proportions  in  the  gases  in 
such  a  pneumothorax:  CO2,  1.76  per  cent.;  O,  18.93  per  cent.;  and  79.31  per  cent. 
N.  Here  the  proportion  of  CO2  is  even  a  little  less  than  in  ordinary  expired  air, 
which  contains  3.3-3.5  per  cent.  When  air  .enters  a  closed  pleural  cavity  and  no 
effusion  follows,  it  is  slowly  absorbed  until  a  mixture  of  about  90  per  cent.  N,  4  per 
cent.  O  and  6  per  cent.  CO2  results;  but  if  there  is  a  serous  effusion  the  oxygen 
disappears  nearly  or  quite  completely  (Tobiesen).*^  In  a  seropneumothorax 
Ewald  found  8.13  per  cent,  of  CO2,  1.26  per  cent,  of  O,  and  90.61  per  cent,  of  N, 
which  is  quite  similar  to  the  proportions  of  the  gases  in  dry  pneumothorax.  Puru- 
lent pneumothorax  generally  shows  more  CO2  than  the  serous  form,  the  average  in 
the  former  being  15-20  per  cent.,  in  the  latter  7.5-11.5  per  cent.  The  average  of 
the  analyses  in  six  cases  of  pyopneumothorax  is  given  by  Ewald  as  18.13  per  cent. 
CO2,  2.6  per  cent.  O,  and  79.81  per  cent.  N.  In  open  pyopneumothorax  the  gas 
approaches  more  closely  the  composition  of  air,  but  usually  shows  a  slight  excess  of 
C()2;  it  is  thus  possible  by  a  determination  of  the  carbon  dio.xide  to  determine 
quite  accurately  whether  a  given  pneumothorax  is  in  communication  with  the 
outside  air.  The  transformation  of  a  purulent  into  a  putrid  pneumothorax  is 
accompanied  by  an  increase  of  CO3,  even  as  high  as  40  per  cent,  having  been  found. 
The  products  of  decomposition  by  the  putrefactive  saprophj'tes  also  are  present, 
one  analysis  having  shown  4.3  per  cent,  of  hydrogen,  6.25  per  cent,  of  methane,  and 
traces  of  hydrogen  sulphide. 

Infection  of  a  pleural  effusion  by  gas-producing  organisms  may  also  convert  it 
into  a  pneumothorax,  although  this  is  not  a  common  occurrence.     The  gases  then 

82  Complete  literature  and  resume  given  by  Clemens,  in  Ott's  "Chem.  Path 
der  Tuberculose,"  Berlin,  1903,  p.  406. 

"  Beitr.,  z.  lOin.  d.  Tuberk.,  1911  (19),  451;  1911  (21),  109;  Deut.  Arch.  klin. 
Med.,  1914  (115),  399. 


366  EDEMA 

present  are  the  same  as  the  organisms  produce  in  similar  culture-media,  modified 
somewhat  by  absorption.  The  anaerobic  gas-producing  organisms  have  been 
found  as  the  cause  of  such  gaseous  accumulations;  it  is  questionable  if  the  ordinary 
pathogenic  organisms  can  cause  a  pneumothorax,  since  they  are  for  the  most  part 
not  capable  of  producing  gas.  The  colon  bacillus  produces  gas  in  sugar-containing 
media,  but  the  amount  of  sugar  in  the  pathological  exudates  is  too  small  to'yield 
any  considerable  amount  of  gas;  an  exception  is  the  pleural  effusion  in  diabetes,  and 
pneumothorax  from  infection  of  the  pleural  effusion  in  a  diabetic  by  B.  coli  has 
been  reported.  Complete  quantitative  analyses  of  the  gas  in  this  form  of  pneu- 
mothorax seem  not  to  have  been  made,  but  May  found  about  20  per  cent,  of  CO2. 
The  combustibility  of  the  gas  has  i^frequently  been  noted,  and  is  probably  dueto 
hydrogen  and  methane. 


CHAPTER  XV 

RETROGRESSIVE   CHANGES  (NECROSIS,  GANGRENE,  RIGOR 
MORTIS,  PARENCHYMATOUS  DEGENERATION) 

NECROSIS 

We  recognize  that  a  cell  is  alive  through  its  reproducing,  func- 
tioning, and  its  taking  on  and  utilizing  nutritive  substances;  yet 
at  the  same  time  we  appreciate  that  a  cell  may  do  none  of  these  things 
and  still  be  alive.  For  example,  a  bacterial  spore  is  quite  inert  physi- 
cally, and  exhibits  no  chemical  activity,  yet  it  is  by  no  means  dead, 
since  it  still  possesses  the  latent  power  to  assume  again  an  active  exist- 
ence under  suitable  conditions.  In  pathological  conditions  we  are 
accustomed  to  recognize  the  fact  that  a  cell  is  dead  by  certain  altera- 
tions in  its  structural  appearance,  particularly  disintegrative  changes 
in  the  nucleus;  but  this  is  exactly  equivalent  to  recognizing  that  an 
animal  is  dead  by  the  appearance  of  postmortem  decomposition,  for 
most  of  the  characteristic  histological  changes  of  necrosis  are  merely 
postmortem  changes  in  the  cell.  A  cell  may  be  dead  and  show  ab- 
solutely none  of  these  microscopic  disintegrative  changes,  either  because 
it  has  not  been  dead  long  enough  for  them  to  have  taken  place,  or 
because  the  changes  have  been  prevented  by  some  means,  just  as  we 
can  prevent  the  appearance  of  postmortem  decomposition  by  embalm- 
ing. For  example,  if  we  examine  microscopically  the  mucous  mem- 
brane of  the  stomach  of  a  person  who  has  died  immediately  after 
taking  a  large  quantity  of  carbolic  acid,  although  to  the  naked  eye  this 
mucous  membrane  is  hard,  white,  and  definitely  necrotic,  yet  we  find 
the  histological  picture  presented  by  the  cells  almost  absolutely  un- 
changed from  the  normal.  The  cells  are  dead,  but  they  have  been  so 
"fixed "  that  postmortem  changes  could  not  affect  their  structure.  All 
cells  examined  by  ordinary  histological  methods  are,  of  course,  dead — 
killed  by  the  fixing  agents  outside  of  the  body,  in  the  same  way  that 
the  carbolic  acid  fixes  them  within  the  body.  It  is  evident,  therefore, 
that  it  may  be  very  difficult  to  determine  always  whether  a  cell  is 
dead  or  not.  Part  of  the  difficulty,  perhaps,  hes  in  our  failure  to 
appreciate  that  not  all  parts  of  a  cell  die  at  the  same  time;  i.  e.,  the 
different  chemical  processes  of  the  cell  depend  on  its  different  intracell- 
ular enzymes,  and  these  are  not  necessarily  destroyed  alike  by  the 
same  agents.  Even  considerable  respiratory  activity  may  be  ex- 
hibited by  cells  that  have  been  killed.^" 

We  recognize  that  after  an  animal  is  dead  as  a  whole  the  various 
cells  of  its  body  do  not  die  for  some  time  as  shown  by  the  following 

i^See  Haas,  Bot.  Gazette,  1919  (67),  347. 

367 


368  RETROGRESSIVE  CHANGES 

examples:  (1)  We  can  cause  the  heart  to  beat  for  a  considerable 
period  after  its  removal  from  the  body;  (2)  if  we  perfuse  a  mixture 
of  glycocoll  and  benzoic  acid  through  the  kidney  of  a  recentl}'  killed 
animal,  synthesis  of  these  substances  into  hippuric  acid  will  occur; 
and  (3)  the  epithelium  of  the  skin  can  be  removed  from  the  body  of 
an  animal  long  after  death  and  transplanted  successfully  on  another 
animal.  So,  too,  in  ordinary  cell  death  (necrobiosis)  not  all  the 
enzymes  are  destroyed  together.  When  all  are  destroj'-ed  at  once, 
as  by  strong  chemicals  or  by  heat,  the  customary  disintegrative 
changes  do  not  take  place.  If,  however,  not  all  the  enzymes  are 
thrown  out  of  function,  then  the  others  may  be  able  to  act,  producing 
the  disintegrative  changes  by  which  histologists  ordinarily  recognize 
cell  death.  These  disintegrative  changes  are,  for  the  most  part,  ap- 
parently brought  about  by  the  intracellular  proteases,  that  is,  through 
autolysis.  This  may  be  shown  as  follows:^  If  we  take  two  pieces 
of  fresh  normal  tissues  from  an  animal,  and  in  one  kill  the  enzymes 
by  heating  to  100°  C,  then  implant  both  aseptically  into  the  abdom- 
inal cavity  of  an  animal  of  the  same  species,  it  will  be  found  that 
the  changes  that  follow  in  the  two  will  be  very  unlike.  In  the  un- 
heated  tissue  nuclear  changes  soon  occur,  so  that  they  lose  their  ca- 
pacity for  taking  up  basic  stains,  the  cytoplasm  becomes  granular 
and  fragmented,  the  tissue  becomes  friable  so  that  it  is  difficult  to 
secure  good  sections,  and  the  changes  are  in  general  similar  to  those 
seen  in  areas  of  necrosis.  The  boiled  tissue,  on  the  other  hand, 
retains  its  capacity  for  nuclear  staining  for  months,  except  at  the 
periphery,  where  it  is  slowly  attacked  by  leucocytes  and  the  enzymes 
of  the  blood  plasma.  Therefore  it  would  seem  that  the  characteristic 
changes  of  necrosis  depend  chiefly  upon  the  intracellular  enzymes, 
rather  than  upon  the  infiltrating  plasma  as  Weigert^  and  other  early 
writers  imagined.  In  areas  of  anemic  necrosis  (see  "Infarcts") 
we  have  another  case,  in  which  the  oxidizing  enzymes  are  thrown 
out  of  function  through  lack  of  oxygen,  while  the  other  enzymes  are, 
presumably,  at  first  unaffected.  From  studies  of  infarcts  it  would 
seem  that  the  intracellular  proteases  bring  about  the  subsequent 
nuclear  and  cytoplasmic  alterations,  but  that  the  eventual  digestion 
of  the  area  is  accomplished  by  the  invading  leucocytes  working  slowly 
inward  from  the  periphery.  Apparently  when  the  supply  of  materials 
from  outside  ceases,  and  when  the  oxidation  processes  of  the  cells  no 
longer  accomplish  necessary  steps  of  synthetic  reactions  or  destroy 
products  of  protein  catabolism,  the  proteases  continue  to  split  proteins 
without  the  balancing  by  the  above-mentioned  factors,  with  a  resulting 
disintegration  of  the  cells. 

Karyolysis  and  karyorrhexis  are,"then,  the    result  of  an  autolytic 
process,  which  is  perhaps  due  to  intracellular  proteases  that  act  spe- 

1  Wells,  Jour.  Med.  Research,  1906,  (15),' 149. 

2  (Jcnt.  f.  Path.,  1891  (2),  785. 


NECROSIS  369 

cifically  on  nucleoproteins,  and  w  hich  may  l)e  designated  as  nucleases.^ 
Nuclear  staining  by  the  usual  methods  depends  upon  an  affinity  of  the 
acid  nucleoproteins  (in  which  the  nucleic  acid  is  not  completely 
saturated  by  proteins)  for  basic  dyes.  Presumably  in  karyolysis  the 
first  step  consists  in  a  splitting  of  the  nucleoprotein  of  the  chromatin 
into  nucleic  acid  and  protein;  this  can  be  accomplished,  according 
to  Sachs,  by  the  ordinary  trypsin,  and  presumably,  therefore,  by  the 
trypsin-like  enzymes  of  the  cell.  Corresponding  with  this  change 
we  should  expect  the  free  nucleic  acid  to  give  an  intense  staining 
with  basic  stains,  and  this  has  frequently  been  described  by  those 
who  have  studied  the  cytological  changes  in  anemic  necrosis,^  and 
called  pycnosis.  As  supporting  this  view  still  further  may  be  quoted 
Arnheim's^  observation  that  in  alkaline  solutions  the  nucleus  soon 
stains  diffusely  and  weakly,  and  not  at  all  after  twelve  to  eighteen 
hours;  this  is  to  he  explained  by  the  fact  that  nucleic  acid  is  both 
dissolved  and  neutralized  by  alkaline  solutions.  Acids  developed  in 
injured  cells  may,  by  combining  with  the  basic  elements  of  the  nu- 
cleoproteins, render  them  still  more  acid  and  highly  basophilic;  thus, 
in  muscles  showing  waxy  degeneration  from  accumulation  of  lactic 
acid  the  muscle  nuclei  will  be  found  pycnotio  (see  waxy  degenera- 
tion). After  the  nucleic  acid  has  been  freed  from  the  protein  by 
the  autolytic  enzymes,  it  is  still  further  decomposed  by  the  "nu- 
clease" or  similar  intracellular  enzymes  that  have  the  property  of 
splitting  nucleic  acid  into  the  purine  bases  that  compose  it — cor- 
responding with  this  change  the  hyperchromatio  nucleus  loses  its 
affinity  for  stains,  and  karyolysis  is  complete.  When  extensive  ne- 
crosis occurs  there  will  result,  therefore,  an  increased  elimination 
of  purines,  as  was  found  by  Jackson  and  Pearce^  in  animals  with 
severe  hepatic  necrosis  from  hemotoxic  serum. 

A  careful  analytical  study  of  the  changes  taking  place  in  the  autolyzing  spleen, 
for  the  purpose  of  correlating  the  chemical  and  microscopical  changes,  has  been 
made  by  Corper,^  which  corroborates  the  interpretation  of  necrosis  advanced 
above.  He  found  that  during  the  stage  when  pycnosis  is  the  chief  feature  there 
is  no  appreciable  change  in  the  nucleus;  that  is,  the  nucleic  acid  has  not  been 
split  into  free  purines  and  the  rest  of  its  components;  at  this  stage  but  little 
change  has  occurred  in  the  lecithin,  and  a  very  slight  amount  of  proteolysis  is 
demonstrable.  During  the  stage  of  karyorrhexis  and  karyolysis  the  most  active 
disintegration  is  taking  place,  alDOut  one-fourth  of  the  nucleic  acid  becoming  dis- 
integrated by  the  time  all  nuclear  structures  have  disappeared;  in  the  same 
period  nearly  half  the  lecithin  (phosphatids)  is  hydrolyzed,  while  about  one- 
fourth  the  coagulable  protein  has  been  hydrolyzed  into  non-coagulable  compounds. 
After  this  stage  the  changes  are  very  slow.  It  is  somewhat  surprising  to  find 
that  when  no  vestige  of  nuclear  substance  remains  in  stainable  form,  there  still 
remains  three-fourths  of  the  nucleic  acid  in  an  intact  condition.     Corper  publishes 

^  See  Purine  Metabolism,  Chap,  xxiii. 

■*  Schmaus  and  Albrecht,  Virchow's  Arch.,  1895  (138),  supp.,  p.  1;  Ergeb.  allg. 
Pathol.,  1896  (3),  486  (literature). 
*  Virchow's  Arch.,  1890  (120),  367. 
«  Jour.  Exper.  Med.,  1907  (9),  569. 
'  Jour.  Exper.  Med.,  1912  (15),  429. 

24 


370  RETROGRESSIVE  CHANGES 

a  series  of  plates,  together  with  the  chemical  details'  thus  establishing  a  standard 
whereby  the  histological  changes  can  be  interpreted  in  terms  of  the  chemical 
changes  which  cause  them. 

Autolysis  of  asepfcically  preserved  tissues  outside  the  body  is 
much  more  rapid  than  is  the  autolysis  of  infarcts  and  similar  aseptic 
necrotic  areas  within  the  body.  This  may  be  due  to  either  or  both  of 
two  factors:^  First,  autolysis  is  much  slower  in  alkaline  than  in  acid 
media;  outside  the  body  autolyzing  tissues  develop  an  acid  reaction 
which  favors  their  autolysis;  within  the  body  this  is  checked  by  the 
plasma.  Second,  the  plasma  contains  inhibiting  substances,  which 
also  may  interfere  with  self -digestion  in  the  body.  In  corroboration 
of  the  above  may  be  recalled  the  fact  that  large  necrotic  areas  show 
autolysis  first  in  the  center,  where  the  alkaline,  antagonistic  body 
fluids  presumably  cause  the  least  effect.  Furthermore,  it  has  been 
found  by  Wells^  that  the  histological  changes  of  autolysis  proceed 
much  faster  in  tissues  placed  in  serum  that  has  been  heated  to  destroy 
the  antibodies  than  in  unheated  serum.  Leucocytes,  as  Opie  has 
shown,  contain  autolytic  enzymes  acting  best  in  an  alkaline  medium, 
hence  they  perform  their  digestive  function  readily  at  the  periphery 
of  necrotic  areas,  and  coagulated  tissue  proteins,  when  acted  upon  by 
body  fluids,  produce  chemotactic  substances  which  attract  leucocytes 
to  dead  areas. ^° 

When  a  cell  dies,  certain  physical  changes  occur  that  are  probably 
of  considerable  importance.  Bechhold  says:  "With  the  occurrence 
of  death,  protoplasm  gelatinizes,  Brownian  movement  of  the  smaller 
particles  ceases,  and  the  structure  of  the  gel  appears  in  the  ultramicro- 
scope  as  a  conglomeration  of  many  reflecting  platelets.  It  makes  a 
substantial  difference  whether  the  protoplasm  slowly  dies  or  is  suddenly 
killed  by  a  fixative  (alcohol,  formalin,  etc.).  In  the  first  instance 
there  is  a  precipitation  (flocculation),  whereas,  in  the  latter  there  is 
a  stiffening;  this  difference  may  be  readily  recognized  under  the 
ultramicroscope." 

The  permeability  of  the  cell  wall  is  almost  immediately  increased, 
so  that  all  diffusible  substances  readily  pass  through,  i.  e.,  its  semi- 
permeable character  is  lost.  This  we  see  particularly  in  plant  cells, 
which  lose  their  turgor  with  their  semipermeability,  and  therefore 
the  plant  wilts.  The  cell  structure  is  also  disintegrated,  and  as  a 
result  coordination  of  the  cell  chemistry  is  at  once  destroyed. ^^  In- 
tracellular enzymes  escape  into  the  blood  from  areas  of  local  death  of 
cells, ^2  or  as  an  agonal  manifestation  in  general  death. ^^     Various 

*  Literature  and  more  complete  discussion  under  "Autolysis." 
9  Jour.  Med.  Research,  190G  (15),  149. 
10  Burger  and  Dold,  Zeit.  Immunitiit.,  1914  (21),  378. 

"  See  V.  Prowazek,  Biol.  Centrbl.,  1909  (29),  291.  Pictet  suggests  that  in 
dead  proteins,  aldehydes  and  amino  radicals  unite  with  one  another  to  form  cyclic 
compounds  (Arch.  sci.  phys.  nat.,  1915  (40),  181). 

12  Mandelbaum,  Munch,  med.  Woch.,  1914  (()1),  461. 

13  Schultz,  Miinch.  med.  Woch.,  1913  (GO),  2512. 


NECROSIS  371 

dyes  which  cannot  penetrate  hvinj;  cells  may  stain  dead  or  dying  cells.'* 
These  changes  depend  on  alterations  in  permeability,  and  as  permea- 
bility determines  electrical  resistance,  Osterhout  has  used  the  resistance 
of  plant  cells  as  an  indicator  of  vitality.  He  finds  that  normal  cells 
have  a  rather  constant  resistance,  which  is  reduced  by  anything  that 
lowers  the  vitality'  of  the  cell,  and  in  direct  proportion  to  the  degree 
of  injur}'  or  loss  of  vitality.'^  The  temperature  coefficient  is  also 
considerablj'  lower  in  dead  than  in  living  tissue. ^^  When  secondarj' 
disintegrative  changes  occur  in  the  protoplasm,  with  the  formation  of 
many  small  molecules  from  the  large  molecules  of  the  cell,  both  osmo- 
tic pressure  and  electrical  conductivity  increase  rapidly.  Changes 
in  the  permeability  of  cell  protoplasm,  however,  may  be  of  considerable 
degree  without  necessarily^  indicating  serious  injury  of  the  cells  (Oster- 
hout).^^  Death  is  accompanied  by  changes  of  the  character  of  a 
monomolecular  reaction,  which  is  continually  going  on  and  w'hich  is 
accelerated  by  the  toxic  agent. '^  Up  to  a  certain  point  the  reaction 
seems  to  be  reversible. 

A  principle  of  colloid  chemistry,  the  alteration  of  colloids  with  time,  has  an 
interesting  bearing  on  the  question  of  aging  and  natural  death  of  tissues.'*"  It 
is  characteristic  of  colloidal  solutions  (which,  of  course,  is  what  cells  are),  that 
they  continuously  change  in  their  properties,  the  change  being  generally  in  the 
direction  of  aggregation  of  the  disperse  colloidal  particles,  with  a  resulting  ten- 
dency to  precipitation  or  coagulation;  the  gels  tend  to  decrease  in  elasticity  and 
to  become  more  turbid,  associated  with  which  are  alterations  in  their  perme- 
ability to  crystalloids.  A  gelatin  mass  possesses  its  maximum  elasticity  three 
or  four  hours  after  it  is  first  formed;  and  crystalloids  penetrate  fresh,  quickly- 
formed  gels  at  first  more  rapidly  than  later.  As  Bechhold  says,  we  can  imagine 
(1)  a  relation  of  such  facts  to  the  greater  elasticity  of  young  tissues;  (2)  to  a  pre- 
sumably greater  permeability  for  crystalloids  and  hence  more  rapid  metabolism; 
(3)  to  the  decreasing  water  of  the  tissue  with  age  (94  per  cent,  of  water  in  the 
fetus  of  three  months,  69-66  per  cent,  at  birth,  and  58  per  cent,  in  adults);  (4) 
to  the  demonstrated  greater  permeability  of  young  nerve  tissues  for  vital  stains, 
etc.  "In  general  we  can  say  that  the  tissue  colloids  decrease  in  their  water  affinity 
(Quellbarkeit)  both  in  animal  organisms,  which  become  poorer  in  water  with  age, 
and  in  plants,  as  shown  by  the  hardening  of  older  plant  tissues."  The  bearing 
of  these  principles  on  the  problem  of  senility  and  degeneration  of  elastic  tissue, 
regeneration  and  many  other  subjects  is  obvious. 

Causes  of  Necrosis 

Anemia. — After  the  cutting  off  of  blood-supply,  cells  soon  undergo 
morphological  changes  that  we  recognize  as  indicating  their  death,  and 
after  a  time  they  also  become  incapable  of  returning  to  their  normal 
condition  when  the  blood-supply  is  re-established,  probably  because 
of  these  structural  changes.     In  just  what  way  lack  of  nourishment 

"  See  Steckelmacher,  Beitr.  path.  Anat.,  1913  (57),  314. 

15  See  Science,  1914  (40),  488. 

1^  Galeotti's  earher  observations  with  animal  tissues  (Zeit.  f.  Biol.,  1903  (45), 
65)  do  not  harmonize  with  Osterhout'fe  results,  and  Galeotti's  idea  that  there  is  a 
special  degree  of  ionization  characteristic  of  living  cells  is  not  established. 

1^  Botan.  Gaz.,  1915  (59),  242. 

18  Osterhout,  Jour.  Biol.  Chem.,  1917  (31;,  585. 

18"  See  H.  Bechhold,  "Die  Kolloide  in  Biologic  und  Medizin,"  Dresden,  1912, 
„•  65. 


"372  RETROGRESSIVE  CHANGES 

causes  death  has  not  been  determined,  but,  as  has  been  before 
suggested,  it  seems  probable  that  it  is  because  cataboUc  processes  are 
no  longer  balanced  by  anabolic  processes,  and  with  these  latter  oxi- 
dizing enzymes  seem  to  be  inseparab'y  associated  as  far  as  our  pres- 
ent knowledge  shows  us.  That  the  loss  of  oxygen  alone,  with  other 
materials  presumably  supplied  to  the  cells  in  adequate  amount,  may 
cause  necrosis,  is  shown  by  the  presence  of  marked  hepatic  necrosis 
in  animals  kept  a  week  in  atmospheres  extremely  low  in  oxygen  (5-9 
per  cent.)-^^  The  nature  of  the  chemical  changes  taking  place  in  a 
cell  when  oxj^gen  is  deficient  must  be  very  different  from  the  normal 
changes,  and  hence  abnormal  toxic  substances  may  accumulate,  e.  g., 
excessive  amounts  of  organic  acids.  Were  it  not  that  the  proteolytic 
enzymes  continue  in  action  after  nutrition  is  shut  off,  the  cells  might 
remain  in  a  completely  unaltered  condition  for  an  indefinite  period, 
and  capable  of  resuming  their  function  when  nourishment  is  again 
supplied,  which  is  decidedly  contrary  to  the  facts.  (The  general 
features  of  anemic  necrosis  have  been  already  discussed  in  the  pre- 
ceding paragraphs,  and  also  under  the  subject  of  infarction.) 

Thermic  Alterations. — These  have  been  studied  particularly  in 
connection  with  the  cells  of  the  lower  organisms.^*'  While  some  uni- 
cellular organisms  can  survive  a  temperature  of  69°,  most  of  them 
are  killed  at  from  40°-45°.  For  the  great  majority  of  me+azoa  the 
maximum  temperature  lies  below  45°,  and  in  the  case  of  marine 
species  below  40°.^^  The  heating  is  accompanied  by  the  appearance 
of  granules  in  the  cytoplasm,  which  become  larger  until  the  condi- 
tion of  "heat  rigor"  sets  in.  Kiihne,  in  1864,  showed  that  in  muscle 
cells,  at  least,  there  is  contained  a  protein  which  becomes  turbid 
through  partial  coagulation  at  40°,  and  Halliburton-^  has  found 
that  in  nearly  all  tissues  are  globulins  coagulating  at  from  45°-50°; 
it  is  probable,  therefore,  that  the  granules  formed  in  heated  cells  are 
produced  through  coagulation  of  these  proteins.  The  importance  of 
this  coagulation  in  determining  death  is  not  yet  fully  established, 
but  it  would  seem  to  be  very  great.  Halliburton  has  observed  that 
in  both  muscles  and  nerves  to  which  heat  is  applied,  contractions 
occur  at  various  temperatures,  corresponding  exactly  with  the  tem- 
peratures at  which  the  several  varieties  of  the  proteins  of  the  cells 
coagulate.  Furthermore,  Mott-^  has  found  that  the  temperature 
that  is  immediatel}''  fatal  to  mammals  (47°)  is  exactly  the  same  as 
the   coagulating  temperature  of  the  lowest   coagulating  protein  of 

19  Martin,  Loevenhart  and  Bunting,  Jour.  Exp.  Med.,  191S  (27),   399. 

^"Literature,  see  Davenport,  "Experimental  Morphology,"  New  York,  1S97; 
Schmaus  and  Albrecht,  Ergebnisse  dcr  Pathol.,  1890  (."3,  Abt.  1),  470. 

^'  Tlie  adaptation  of  animal  cells  to  high  tcniperaturos  i.s  an  interesting  topic, 
especially  in  view  of  such  results  as  those  of  Dallingor,  who,  by  raising  the  tem- 
perature gradually  during  several  years,  caused  flagollatn,  with  a  normal  maxinuuu 
of  about  21°  -23°  to  become  capable  of  living  at  70°  (see  Davenpotr.). 

"  "Biochemistry  of  Muscle  and  Nerve,"  Phila.,  1904. 

2^  Quoted  by  Halliburton. 


NECROSIS  373 

norve-cells.  This  fact  is  undoubtedly  of  great  practical  importance 
in  causing  death  from  fever,  for  although  47°  C.  (117°  F.)  is  prob- 
ably never  reached  in  man,  yet  application  of  much  lower  tempera- 
tures, even  42°  (108°  F.),  for  a  few  hours  will  cause  coagulation  of 
these  proteins  (all  proteins  coagulate  at  less  than  their  ordinary 
coagulation  point  if  the  heating  is  continued  for  a  long  time).  It 
would  seem  from  the  above  observation  that  heat  may  cause  cell  death 
through  coagulation  of  the  proteins.  Whether  the  cell  death  is  in 
any  way  dependent  upon  destruction  of  the  enzymes  by  heat  has  not 
l)een  ascertained;  but  as  most  enzymes  are  not  destroyed  nmch  be- 
low 60°-70°,  it  seems  impro})able  that  they  are  greatly  injured  at 
the  temperatures  at  which  cells  are  killed.  It  is  possible,  however, 
that  under  the  conditions  in  which  enzymes  exist  in  the  cell  they 
may  be  more  susceptible  to  heat  than  under  other  conditions.  Just 
how  coagulation  of  cell  globulins  can  determine  the  death  of  a  cell 
is  difficult  to  understand,  unless  the  physical  conditions  of  the  cell 
are  greatly  altered  thereby.  Ordinarily  we  have  in  the  cell  an  equi- 
librium between  colloids  in  solution  and  colloids  in  the  solid  or  gel 
state;  if  the  colloids  are  rendered  insoluble  by  heat,  or  by  any  other 
cause,  so  that  this  equilibrium  is  destroyed,  serious  alterations  in  the 
mechanism  of  all  metabolism  must  result  (Mathews).  Other  chem- 
ical reactions  will  also  have  their  point  of  equihbrium  altered  by 
changes  in  temperature,  and  such  alterations  might  well  have  disas- 
trous results. 

Different  tissues  show  unequal  susceptibility  to  heat.  Werhov- 
sky^^  found  the  blood  most  affected  by  raising  the  temperature  of 
living  animals,  next  the  liver,  kidneys,  and  myocardium  in  order, 
the  other  tissues  being  little  or  not  at  all  structurally  injured.  Ani- 
mals exposed  to  heat  show  a  fall  in  the  leucocyte  count,  followed  by  a 
rise  in  lymphocytes  which  persists;  there  is  an  extensive  degeneration 
of  cells  in  the  spleen  and  lymph  glands,  followed  by  marked  mitotic 
proliferation  in  the  germinal  centers. ^^ 

Cold^^"  is  well  withstood  by  unicellular  forms,  and  relatively  poorly 
by  more  complex  organisms,  particularly  by  those  with  a  highly  de- 
veloped circulatory  system;  this  is  because  individual  cells  are  not 
greatly  affected  by  freezing,  whereas  the  circulatory  channels  are 
readily  blocked  by  this  cause.  Bacterial  cells  are  not  killed  by  ex- 
posure for  long  periods  to  the  temperature  of  liquid  air^^  (—190°). 
Reduction  of  the  temperature  of  plant  cells  to  —13°  may  result  in 
a  granular  transformation  of  the  cytoplasm,  often  with  rather  seri- 
ous structural  alterations.  Cytoplasm  seems  to  be  more  affected  than 
the   nucleus,  for  mitosis  may  occur   slowly  in  plant  cells  at  —8°, 

2*  Ziegler's  Beitr.,  18P5  (18),  72. 

"  Murphy  and  Sturm,  Jour.  Exp.  Med.,  1919  (29),-  1. 

-^*  Sy:^temic  rffectsof  cold  reviewed  by  Foord,  Jour.  Infect.  Dis.,  1918(23),  159. 
2  MacFadyrn,  Lan  et,  1900  (i),  849. 


374  RETROGRESSIVE  CHANGES 

and  Uschinsky^^  noted  that  in  animal  tissues  the  nuclei  were  less  af- 
fected b}^  cold  than  the  cytoplasm.  Blood  seems  little  affected  by 
freezing  temperature,  for  du  Cornu  found  that  dog's  blood  kept  on 
ice  for  five  to  ten  days  could  be  employed  for  transfusion  without 
causing  hemoglobinuria.  Grawitz  saw  motion  persist  in  human  cili- 
ated epithelium  kept  for  seven  to  nine  days  on  ice.  Cihated  epi- 
thelium from  the  mouth  of  the  frog  may  survive  cooling  to  —90° 
and  frog  eggs  are  not  killed  by  —60°.  In  many  cells,  however,  the 
physical  changes  produced  by  freezing,  and  also  by  the  subsequent 
thawing,  are  sufficient  to  render  them  incapable  of  further  existence.'^* 
Cells  devoid  of  or  poor  in  water  cannot  be  killed  by  freezing,  hence 
it  is  probable  that  the  currents  set  up  about  the  crystals  of  ice  in 
thawing,  as  well  as  the  rapid  contraction  and  expansion  under  the 
influence  of  the  cold  and  the  ice  formation,  are  the  cause  of  the  effects 
of  freezing,  which,  therefore,  are  not  dependent  upon  chemical,  but 
upon  physical,  alterations. 

In  the  case  of  warm-blooded  animals,  the  gangrene  following  freez- 
ing depends  not  so  much  upon  the  freezing  of  the  cells  themselves  as 
upon  the  formation  of  hyalin  thrombi  in  the  injured  vessels  (v.  Reck-j 
linghausen,  Hodara).^^  Kriege^''  found  that  if  the  freezing  is  transi- 
tory, the  thrombi  may  again  disappear;  if  over  two  hours  in  duration, 
they  are  persistent.  Rischpler,^^  however,  considers  that  cell  death 
is  due  primarily  to  the  effect  of  the  cold  upon  the  cells,  and  Lake^- 
found  that  for  both  isolated  cells  in  culture  and  living  tissues  with 
intact  blood  supply,  deai.b  occurred  at  —6°  C,  this  being  the  tempera- 
ture at  which  protoplasm  freezes.  On  the  other  hand,  Steckel- 
macher^^"  found  that  freezing  of  liver  tissue  produced  the  same  changes 
as  ligation  of  the  hepatic  artery,  i.  e.,  increased  permeabiHty  of  the 
cell  wall  followed  by  similar  changes  in  the  nucleus,  suggesting  that 
the  changes  produced  by  freezing  depend  on  the  vascular  changes. 

Light.^'^ — ^Light  may  affect  tissues  seriously,  apart  from  the  effects 
of  accompanying  heat,  although  the  experiments  of  Aron^^  indicate 
that  insolation  does  not  depend  on  the  light  raj^s,  but  solely  on  the 
heat.  In  the  treatment  of  lupus  by  the  Finsen  method  with  concen- 
trated light  rays,  the  action  is  largely  a  stimulating  one,  but  associ- 
ated with  or  subsequent  to  a  certain  degree  of  cell  injury.     Ogneff^^ 

"  Ziegler's  Beitr.,  1893  (12),  115. 

^8  In  plant  cells  it  is  the  freezing  and  not  the  thawing  that  causes  the  harm 
(Maximow,  Berichte  Deut.  Bot.  Gesell.,  1912  (30),  50-4). 

29  Miinch.  med.  Woch.,  1896  (43),  341. 

30  Virchow's  Arcli.,  1889  (116),  64. 
"  Ziegler's  Beitr.,  1900  (28),  541. 
32  Lancet,  Oct.  13,  1917. 

32"  Beitr.  path.  Anat.,  1913  (57),  314. 

"  Review  by  Bering,  Ergeb.  allg.  Pathol.,  1914,  Abt.  1  (17),  790.  See  dis- 
cussion of  the  principles  of  the  action  of  light  on  tissues  by  Bovie,  Anier.  Jour. 
Tropical  Dis.,  1915  (2),  506. 

•"•  PliiU])i)ine  Jour.  Sci.,  B,  1911  (6),  101. 

a'  Pfiiiger's  Arch.,  1896  (63),  209. 


NECROSIS  375 

fouiul  that  inodcmte  action  of  electric  light,  rich  in  violet  and  ultra- 
violet raj's,  causes  mitotic  cell  division;  if  the  action  is  stronger,  the 
cells  undergo  amitotic  division  and  then  become  necrotic.  Blue  rays 
have  but  slight  cytotoxic  action,  and  rays  further  towards  the  red  end 
of  the  spectrum  are  without  demonstrable  cffec '..  Light  baths  are 
said  by  Oerum"'  to  increase  greatly  the  quantity  of  corpuscles  and  hemo- 
globin, while  residence  in  the  dark  reduces  these  elements.  The  de- 
struction of  bacteria  by  light  is  a  well-known  phenomenon, ^^  but  it 
has  been  suggested  that  their  destruction  depends  rather  upon  the 
action  of  substances  produced  in  the  culture-medium  under  the  influ- 
ence of  light  than  upon  the  effect  of  the  hght  upon  the  bacterial  cells 
themselves.  In  view  of  the  fact  that  enzymes  and  antibodies  in  solu- 
tion are  quite  readily  weakened  or  destroyed  by  the  action  of  light,  it 
is  possible  that  intracellular  enzymes  may  be  similarly  destroyed  by 
light,  with  resulting  cell  death.  However,  in  the  case  of  bacteria,  at 
least,  the  effects  of  light  seem  to  depend  upon  oxidation  processes,  for 
in  the  absence  of  oxygen,  bacteria  are  not  seriously  injured  by  light, 
and  D'Arc}^  and  Hardy^*  found  that  "active  oxygen"  is  formed  by 
the  same  portion  of  the  spectrum  that  is  most  active  in  destroying 
bacteria. ^^  Light  may  also  alter  the  solubility  of  cell  proteins,  espe- 
cially in  the  presence  of  various  organic  and  inorganic  substances  that 
act  as  sensitizers,  such  as  silica"!  es,  sugar,  lactic  acid  or  urea.^*^  In 
this  may  lie  the  cause  of  cataract,  especially  diabetic  cataract. 

The  general  effect  of  light  acting  on  organic  substances  present  in 
plant  and  animal  cells,  is  to  produce  from  carbonyl-containing  materi- 
als aldehyde  or  ketone  compounds,  whose  reactivity  and  availability 
for  important  synthetic  changes  are  conspicuous  (Neuberg).*^ 
Whether  oxidative  processes  are  the  cause  of  death  in  animal  cells  is 
not  known,  but  we  are  familiar  with  many  chemical  reactions  of  vari- 
ous sorts  that  are  initiated  or  checked  by  the  action  of  light. ^^  Thus, 
bilirubin  is  oxidized  into  biliverdin,  when  acted  upon  by  sunlight, 
even  when  not  in  contact  with  air;  many  vegetable  oils  are  oxidized 
by  sunlight,  and  it  is  probable  that  the  oxidizing  action  of  light  upon 
organic  compounds  is  of  wide-spread  occurrence.  It  is,  therefore, 
quite  possible  that  such  oxidative  changes  may  be  the  cause  of  necrosis 
produced  by  the  action  of  light  rays,  especially  as  Bering'*^  has  found 
that  chemically  active  light  rays  have  a  direct  action  on  oxidizing 
enzymes. 

36  Pfluger's  Arch.,  1906  (114),  1. 

3'  Literature  given  by  Wiesner,  Arch.  f«Hyg.,  1907  (61),  1. 

38  Jour,  of  Physiol.,  1895  (17),  390. 

33  See  also  Agulhon,  who  found  that  ultraviolet  rays  may  attack  enzymes  to 
some  extent  in  the  absence  of  oxygen  (Ann.  Inst.  Pasteur.,  1912  (26),  38). 

"Schanz,  Biochem.  Zeit.,  1915  (71),  406;  Arch.  Ophthal.,  1918  (96),  172; 
Burge,  Amer.  Jour.  Physiol.,  1916  (39),  335;  Neuberg  and  Schwarz,  Berl.  klin. 
Woch^  1917  (54),  84. 

"  Biochem.  Jour.,  1908  (13),  305. 

"See  Davenport,  "Experimental  Morphology,"  1897,  p.  162. 

"  Miinch.  med.  Woch.,  1912  (59),  2795. 


376  RETROGRESSIVE  CHANGES 

It  is  yery  probable  that  not  all  of  the  effects  of  exposure  to  the  sun 
depend  upon  the  heat  rays,  for  there  is  evidence  that  the  hght  rays 
may  also  produce  effects.  This  is  definitely  true  in  the  case  of  indi- 
viduals or  animals  with  certain  pigments  in  their  blood,  notablj^ 
hematoporphyrin  (q.  v.).  In  them,  not  only  may  skin  eruptions  re- 
sult from  relatively  small  exposure  to  light,  but  mice  may  be  so  sen- 
sitized that  a  few  moments  of  exposure  to  light  is  fatal. ^*  Artificial 
fluorescent  substances,  such  as  eosin,  also  sensitize  tissues  and  proteins 
to  light. ^^  Normal  blood  absorbs  light  rays  in  large  amounts,  as 
Finsen  showed,  and  it  is  quite  possible  that  changes  in  the  chemistry 
of  the  blood  result  from  the  light  rays.  Exposure  to  the  sun  may 
cause  a  general  leucocytosis  with  relative  lymphocytosis.^^ 

According  to  HerteP^  the  ultraviolet  rays  cause  oxygen  to  spUt  off 
the  easily  oxidizable  compounds  of  protoplasm,  and  Bovie*^  found 
that  they  coagulate  proteins;  they  also  have  a  destructive  effect  on 
enzymes, ^^  serum  complement'*^  and  hormones. ^^  However,  Burge,^^ 
found  that  exposure  of  living  cells  to  ultraviolet  radiation  of  sufficient 
intensity  to  kill  the  cells  does  not  decrease  to  any  appreciable  extent 
the  activity  of  the  intracellular  enzymes;  the  cell  death  he  attributes 
to  coagulation  of  protoplasm.  Harris  and  Hoyt^^  advance  evidence 
that  the  susceptibility  of  protoplasm  to  ultraviolet  light  is  conditioned 
by  selective  absorption  of  the  toxic  rays  by  the  aromatic  amino-acids 
of  the  proteins.  Toxins  are  reduced  in  activity  by  ultraviolet  raj^s.^' 
X=rays^^  stimulate  cell  growth  when  applied  in  small  amounts,^^ 
but  larger  amounts  produce  necrosis,  which  is  peculiar  in  that  an  in- 
terval of  several  days,  or  even  weeks,  may  elapse  after  the  exposure 
before  the  necrosis  manifests  itself.  Ellis^^  considers  that  the  amount 
of  necrosis  is  out  of  proportion  to  the  changes  in  the  vessels,  which 
some  have  believed  to  be  the  cause  of  x-ray  gangrene,  and  therefore 
that  the  cells  must  be  directly  injured,"  a  view  supported  by  Case- 
mir's^^  experiments  with  plant  cells.     The  extensive  studies  of  the 

"  Hausmann,  Biochem.  Zeit.,  1914  (67),  309. 

**  Full  review  on  photodynamic  action  of  light  by  Sellards,  Jour.  Med.  Res. 
1918  (38),  293. 

«  Aschenheim,  Zeit.  Kinderheilk.,  1913  (9),  87;  Taylor,  Jour.  E.\p.  Med.,  1919 
(29),  41. 

*'  Zeit.  Augenheilk.,  1911  (26),  393. 

*«  Science,  1913  (37),  24;  see  also  Burge,  Amer.  Jour.  Physiol.,  1916  (39),  335. 

"  Brooks,  Jour.  Med.  Res.,  1918  (38),  345. 

60  Burge  et  al,  Amer.  Jour.  Physiol.,  1916  (40),  426. 

"  Amer.  Jour.  Physiol.,  1917  (43),  429. 

"  Science,  1917  (46),  318;  Univ.  Caiif.  Publ.  (Pathol.),  1919  (2),  245. 

"  Ilartoch  et  al,  Zeit.  Immunitat.,  1914  (21),  643. 

**  Full  review  by  Colwell  and  Russ,  "Radium,  X-Rays  and  the  Living  Cell," 
London,  1915.     Also  see  Richards,  Science,  1915  (42),  2S7. 

"  See  Schwarz,  Munch,  med.  Woch.,  1913  (GO),  2165. 

"  Amer.  Jour.  Med.  Sci.,  1903  (125),  85. 

"  Allen  (Jour.  Med.  Research,  1903  (9),  462)  states  that  protozoa  ami  vinegar 
eels  are  killed  by  long  exposure  to  a;-rays,  whereas  plants  are  dooiiledly  stiinulatod 
in  their  growth. 

"  Med.-Naturw.  Arch.,  1910  (2),  423;  rdsuni6  on  a-rays. 


NECROSIS  377 

Hertwigs  show  that  the  chromatin  is  chiefly  affected,  which  presum- 
ably explains  the  fact  that  immature  cells,  and  cells  in  active  division, 
are  more  sensitive  to  a:-rays  than  adult  cells,  and  that  monstrosities 
develop  from  eggs  exposed  to  radiant  energy.  As  far  as  histological 
changes  show,  hard  rays  produce  less  but  (juite  the  same  changes  as 
soft  rays.  That  .r-rays  have  a  marked  effect  on  metabolism  has  been 
abundantly  established.^''  According  to  Musser  and  Edsall,®"  the  ef- 
fect of  :r-rays  upon  metabolism  is  unequalled  by  any  other  therapeu- 
tic agent,  and  is  manifested  by  excessive  elimination  of  the  products 
of  protein  destruction,  which  arise  particular!}'  from  the  lymphatic 
structures. '^'  These  changes  have  been  studied,  therefore,  particu- 
larly in  connection  with  the  treatment  of  leukemia  {g.  v.).  In  con- 
sequence of  the  injury  to  the  blood-forming  tissues,  resistance  to  bac- 
teria is  decreased  (La wen). ^^  The  renal  epithehum  seems  also  to 
suffer  injury  in  some  cases. ""^ 

Exposure  of  the  entire  body  of  animals,  or  large  areas  of  hemato- 
poietic tissue  in  man,  leads  to  profound  changes.  Chief  of  these  are 
destruction  of  lymphoid  cells,  pigmentation  of  the  spleen,  destruction 
of  bone  marrow  cells,  primary  rise  in  poh^morphonuclear  cells  followed 
by  a  fall  to  below  normal,  stead}'  decline  in  lymphocyte  count,  and 
an  increased  resistance  of  the  red  cells  to  radiation. "^^  So  marked  may 
be  the  effect  of  x-rays  on  the  marrow  and  spleen  that  antibody  forma- 
tion is  greatly  depressed  (Hektoen).^^  After  heavy  doses  marked 
metaboHc  changes  occur  which  indicate  a  profound  intoxication,  there 
being  vomiting  and  diarrhoea,  high  non-protein  N  in  the  blood  and  a 
great  increase  in  the  urinary  N  (Hall  and  Whipple).®^  These  authors 
also  observed  necrosis  in  the  intestinal  epi '"helium.  Presumably  these 
reactions  are  similar  to  those  observed  following  superficial  burns,  and 
depend  on  disintegration  of  tissue  proteins  with  production  of  toxic 
substances. 

The  long-continued  action  of  a:-rays  upon  the  skin  has,  in  many 
cases,  led  to  the  formation  of  cancer,  apparently  because  the  pro- 
liferation stimulated  by  the  rays  progresses  until  it  exceeds  normal 

^^  See  Harvey  (Jour.  Path,  and  Bact.,  1908  (12),  548),  concerning  the  effects  of 
x-rays. 

«o  Univ.  Penn.  Med.  Bull.,  1905  (18),  174;  also  Edsall  and  Pemberton,  Amer. 
Jour.  Med.  Sci.,  1907  (133),  426. 

^'  A  peculiar  selective  action  for  the  generative  cells  is  also  shown  by  x-rays, 
which  cause  marked  atrophy  of  the  ovaries  and  testicles.  In  the  latter  it  affects 
chieflv  the  germinative  cells,  sparing  the  cells  of  Leydig.  (See  --Ubere-Schonberg, 
Munch,  med.  Woch.,  1903  (50),  1850;  Frieben,  ibid.,  1903  (50),  2295;  Specht, 
Arch.  f.  Gvn.,  1906  (78),  458;  Thaler,  Deut.  Zeit.  f.  Chir.,  1905  (79),  576;  Reif- 
ferscheid,  Zeit.  f.  Gyn.,  1910  (34),  593. 

62  Mitt.  Grenz.  Med.  u.  Chir.,  1908  (19),  141. 

"  See  Schulz  and  Hoffman,  Deut.  Zeit.  f.  Chir.,  1905  (79),  350;  Warthin,  Amer. 
Jour.  Med.  Sci.,  1907  (133),  736. 

6^  Resume  by  Gudzent,  Strahlentherapie,  1913  (2),  467.  See  also  Taylor  et  al, 
Jour.  Exp.  Med.,  1919  (29),  53. 

"  Jour.  Infect.  Dis.,  1918  (22),  28. 

«  Amer.  Jour.  Med.  Sci.,  1919  (157;,  453. 


378  RETROGRESSIVE  CHANGES 

bounds."    Likewise  leukemia   has   been  observed  several  times  in 
roentgenologists,  presumably  produced  in  the  same  way.®^ 

As  the  metabolic  changes  produced  by  rc-rays  indicate  an  extremely 
high  rate  of  autolysis,  one  may  ascribe  the  effects  either  to  a  stimulat- 
ing effect  of  .T-rays  upon  autolytic  enzjanes,  or  as  Neuberg^^  does,  to 
an  inhibitive  action  of  a;-rays  and  radium  rays  upon  the  other  intra- 
cellular enzymes  without  a  corresponding  deleterious  effect  upon  the 
autolytic  enzymes.^"  This  hypothesis  agrees  with  the  facts  at  hand, 
bui  more  details  concerning  the  effects  of  these  rays  upon  various 
enzymes  are  needed.  The  long  latent  period  before  the  appearance 
of  necrosis  after  exposure  to  x-rays  is  difficult  to  explain^  and  agrees 
rather  with  the  hypothesis  of  slow  proliferative  and  obstructive 
changes  in  the  blood-vessels. 

Radium,  which  shares  with  x-rays  the  power  of  causing  tissue 
necrosis,  does  not  have  so  marked  an  effect  upon  the  blood, '^^  nor  do 
the  ultra-violet  rays  (Linser  and  Helber).''^  In  general,  radium  has 
much  the  same  effect  on  tissues  as  .r-rays,^^  but  seems  rather  to  stimu- 
late the  action  of  most  enzymes  ;^^  autolysis,  however,  is  not  increased 
(Brown). '^^  Radium  partially  destroys  the  growth-promoting  "vit- 
amines"  of  yeast,  which  may  account  for  some  of  its  effects  on  tumors 
(Sugiura  and  Benedict).  Radium  also  causes  severe  skin  lesions  and 
a  general  lymphocytosis  in  those  exposed  to  it  for  long  periods.''^ 
Active  deposit  of  radium  emanation  injected  intravenously  into 
animals  is  highly  toxic,  even  small  doses  causing  fatty  degeneration 
in  the  liver  associated  with  giant  cell  formation  and  hyperchromatic 
nuclei;  larger  doses  cause  multiple  hemorrhages  and  death  with 
severe  enteritis.  Lesions  also  occur  in  the  kidneys,  lungs,  spleen  and 
bone  marrow. '^^"  In  proper  amounts  radium  stimulates  plant  meta- 
bolism (Gager).  Thorium-x  also  attacks  specifically  the  leucocytes, ^^ 
so  that  by  proper  dosage  an  animal  may  be  made  practically  leucocyte- 
s' See  review  by  Wyss.  Beitr.  z.  klin.  Chir.,  1906  (49),  185;  Porter  and  Wol- 
bach.  Jour.  Med.  Res.,  1909  (21),  357. 

«8  See  Jagic  and  Schwarz,  Berl.  klin.  Woch.,  1911  (48),  1220. 

«9  Zeit.  f.  Krebsforschung,  1904  (2),  171;  also  Meyer  and  Bering,  Fortschr. 
Roentgenstrahlen,  1911  (17),  33;  Richards,  Amer.  Jour.  Physiol.,  1914  (36),  400. 

'"  Some  authors  have  believed  certain  of  the  effects  of  .r-rays  to  be  produced 
by  choline  liberated  through  the  decomposition  of  lecithin.  (See  Benjamin  and 
Reuss,  Munch,  med.  Woch.,  1906  (53),  1860.) 

'1  See  Millet  and  Mueller,  Jour.  Cancer  Res.,  1918  (3),  127. 

^2  Deut.  Arch.  kUn.  Med.,  1905  (83),  479. 

'3  Review  by  Guyot,  Cent.  allg.  Path.,  1909  (20),  243;  also  see  Mills,  Lancet 
1910  (179),  462;  Richards,  Science,  1915  (42),  287.  Full  bibliography  by  Sugiura 
and  Benedict,  Jour.  Biol.  Chem.,  1919  (.39),  421. 

7^  Loewenthal,  Berl.  klin.  Woch.,  1910  (47),  287;  Kionlca,  Med.  Klinik,  1911 
(7),  68.5.     Denied  by  Gudzent,  Zeit.  Strahlenther.,  1914  (4),  666. 

'6  T.  R.  Brown,  Arch.  Int.  Med.,  1912  (10),  405. 

"  See  Ordway,  .lour.  Amer.  Med.  Assoc,  1916  (66),  1. 

'*"Bagg,  Jour.  Cancer  Res.,  1920  (5),  1. 

"  See  Plesch  et  al,  Zeit.  exp.  Path.,  1912  (12),  No.  1;  Schweizer,  Miinch.  med. 
Woch.,  1916  (63),  341. 


NECROSIS  379 

frco,^^  whi(;h  has  been  used  for  experimental  studies  on  the  functions 
of  the  leucocytes. 

Electricity. — The  effects  of  the  electric  current  upon  cells  are  de- 
scribed by  Davenport  as  follows:  A  weak  constant  current  causes 
a  centripetal  flowing  of  the  protoplasm  (in  Actinosphaerium) ;  if  the 
current  is  increased  or  long  continued,  the  cytoplasm  of  the  pseudo- 
podia  becomes  varicose,  and  droplets  are  formed  which  soon  burst, 
causing  a  collapse  of  the  protoplasmic  framework.  Finally,  the 
protoplasm  on  the  anode  side  begins  to  disintegrate,  and  the  loose 
particles  move  toward  the  positive  electrode;  eventually  the  cell 
structure  may  be  entirely  destroyed.  A  similar  disintegration  of  the 
anode  side  of  ameba  has  been  observed  by  McClendon,^^  which  he 
attributes  to  anions  which  cannot  pass  through  the  cell  wall,  and 
therefore  accumulate  on  that  side  of  the  organism.  If  an  alter- 
nating current  is  used,  both  anode  and  cathode  sides  of  the  cell  are 
affected.  In  moving  organisms  electric  currents  determine  direction 
of  motion,  even  certain  vertebrates  (tadpoles,  fish)  being  made  to 
orient  themselves  according  to  the  current.  The  nucleus  seems  to  be 
more  susceptible  to  harm  by  electric  currents  than  the  cytoplasm 
(Pfeffer),^"  and  there  seems  to  be  no  oxidation-process  involved  in 
cell  destruction  by  electricity  (as  is  the  case  with  light  rays),  for 
the  effects  are  much  the  same  in  the  absence  of  oxygen  (Klemm). 
Schmaus  and  Albrecht  state  that  the  effect  of  electricity  upon  proto- 
plasm depends  upon  a  loosening  of  the  cohesion  and  a  solution  of  the 
constituents  of  the  cell  (vacuolization),  which  last  is,  perhaps,  due 
to  direct  chemical  alterations.  It  may  be  suggested  that  the  electric 
current  causes  a  migration  of  ions  toward  one  or  the  other  pole  of 
the  cell,  in  this  waj^  separating  the  movable  inorganic  ions  of  the 
ion-protein  compounds  of  the  cell  from  the  immobile  colloidal  pro- 
teins, with  consequent  serious  alterations  in  the  chemistry  of  the  cell. 
Zeit^^  found  that  continuous  currents  kill  bacteria  through  the  pro- 
duction of  antiseptic  substances  in  the  culture-medium,  but  do  not 
harm  them  directly. 

Jellinek^"  has  studied  extensively  the  cause  of  death  after  severe 
electric  shocks,  and  finds  that  there  are  produced  intracerebral  hemor- 
rhages and  degeneration  of  the  nerve-cells,  which  are  sufficient  to 
explain  the  death  of  the  individual  without  having  recourse  to  the 
more  indefinite  idea  of  "shock."  Cunningham^^  considers  fibrillary 
contraction  of  the  heart  as  the  cause  of  death. ^^     Spitzka  and  Ra- 

^^  There  is  no  increase  in  antitrypsin  from  this  leucocyte  destruction  (Rosenow, 
Zeit.  exp.  Med.,  1914  (3),  377). 

'^  Pfitiger's  Arch.,  1911  (140),  271. 

*'' Literature  given  by  Davenport,  "Experimental  Morphology." 

81  Jour.  Amer.  Med.  Assoc,  1601  (37),  1432,  literature. 

82  Virchow's  Arch.,  1902  (170),  56;  Lancet,  1903  (i),  357. 
"  New  York  Med.  Jour,,  1899  (70),  581. 

8^  Full  discussion  by  Jelliffe  in  Peterson  and  Haines'  "Legal  Medicine  and 
Toxicology,"  1903  (1),  245. 


380  RETROGRESSIVE  CHANGES 

dasch^^  find  changes  in  the  brains  of  electrocuted  criminals,  which 
indicate  a  sudden  liberation  of  gas  about  the  blood  vessels,  along  which 
the  current  passes.  The  amperage  seems  to  be  far  more  important  in 
determining  the  effect  of  a  current  than  the  voltage  or  wattage.^® 

Chemicals  cause  cell  death  whenever  they  are  of  such  a  nature  as 
either  to  coagulate  the  cell  proteins  or  to  destroy  its  enzymes.  The 
action  of  such  substances  as  sulphuric  acid,  strong  caustics,  etc., 
hardly  calls  for  explanation.  Phenol  (carbolic  acid)  may  cause  ne- 
crosis and  gangrene  even  when  in  very  dilute  solution;  this  appears 
to  be  due  more  to  the  production  of  hyaline  thrombi  of  agglutinated 
red  corpuscles  in  the  capillaries  than  to  direct  action  upon  the  cells. 
In  some  unpublished  experiments  on  the  subject  of  "carbolic  acid 
gangrene,"  I  found  this  action  of  phenol  very  striking  when  dilute 
solutions  were  placed  on  the  web  of  a  frog's  foot,  under  the  micro- 
scope; as  soon  as  the  solution  penetrated  to  a  capillary,  stasis  with 
fusion  of  the  corpuscles  occurred  in  a  very  few  seconds.  Similar 
results  have  been  obtained  by  Rosenberger.^^  Some  poisons  seem  to 
cause  necrosis  without  destroying  the  autolytic  enzymes,  in  which 
case  the  cells  are  rapidly  digested;  at  least,  such  an  hypothesis  seems 
-to  explain  best  the  changes  seen  in  the  liver  in  chloroform  poisoning, 
acute  yellow  atrophy,  eclampsia,  etc.^*  Not  all  poisons,  b}^  any 
means,  cause  cell  death — tetanus  toxin,  morphine,  and  other  alkaloids 
cause  death  of  the  individual  as  a  whole  without  usually  causing  pri- 
mary necrosis  of  any  of  the  cells.  Cell  death  does  not  necessarily  de- 
pend upon  destruction  of  all  the  cellular  enzymes,  as  has  been  pointed 
out  previously.  Thus,  bacteria  may  be  killed  by  many  chemicals 
which  seem  not  to  affect  their  autolytic  enzymes  seriously.  Any  con- 
siderable excess  of  either  H  or  OH  ions  is  incompatible  with  cell  life, 
and  it  is  possible  that  at  times  the  production  of  acids  within  a  cell 
may  be  sufficient  to  cause  death;^^  e.  g.,  in  the  kidney  in  acute  nephritis 
(M.  H.  Fischer),  or  in  the  muscle  in  waxy  degeneration  ( Wells). ^° 
It  is  quite  probable  that  many  of  the  poisons  act  by  interfering  with 
the  oxidative  capacity  of  the  cells;  this  seems  almost  certain  in  the 
case  of  chloroform  necrosis,  and  even  bacterial  poisons  (diphtheria 
and  typhoid)  were  found  by  Pitini^^  to  decrease  the  oxidizing  power 
of  the  cells. 

The  term,  "protoplasmic  poison,"  has  been  variously  used  and  de- 
fined.    Kunkel  says  that  a  protoplasmic  poison  "is  a  poison  which, 

86  Amer.  Jour.  Med.  Sci.,  1912  (144),  341. 

89  Jellinek,  Wien.  klin.  Woch.,  1913  (26),  1793. 

8'  Verb.  Phys.  Med.  Gesellsch.  z.  Wurzburg,  1900,  vol.  34. 

88  Wells,  Jour.  Amer.  Med.  Assoc,  1906  (46),  341. 

8"  The  partial  protection  afforded  by  a  rich  carbohydrate  diet  against  the 
necrogenic  action  of  chloroform,  phosphorus  and  renal  poisons,  as  observed  by 
Opie  and  Alford  (Jour.  Exp.  Med.,  1915  (21),  1),  may  depend  on  the  antiketogenic 
effect  of  carbohydrates. 

»"  Jour.  Kxp.  Med.,  1909  (11),  1. 

»' Biochem.  Zeit.   1910(253.257. 


NECROSIS  381 

witliout  producing  directly  evident  alterations,  harms  or  kills  all 
living  protoplasmic  structures."  HgCla  is  such  a  poison,  whereas 
H2SO4,  bromine,  and  similar  substances  that  destroy  all  life  through 
their  strong  chemical  action  are  not  included  in  this  category.  The 
j)rotoplasmic  poisons  presumably  act  by  combining  with  one  or  more 
of  the  constituents  of  cell  protoplasm;  e.  g.,  HgCL  i)robably  combines 
with  the  proteins,  chloroform  with  the  cell  lipoids  (physically?).  By 
means  of  his  special  teehnic  Barber^-  is  able  to  introduce  minute 
quantities  of  poisons  into  living  cells  and  observe  their  effect  on  the 
cytoplasm;  HgCl2  is  thus  found  to  be  most  toxic,  while  AS2O3  is 
relatively  inert.  Mathews'-^^  has  shown  that  the  toxicity  of  ions 
depends  on  the  ease  with  which  they  part  with  their  electrical  charges, 
and  the  toxicity  of  a  salt  is  a  function  of  the  sum  of  the  toxicity  of 
the  ions;  hence  the  toxicity  of  a  salt  is  in  inverse  proportion  to  its 
decomposition  tension.  Kunkel  suggests  that  oxalic  acid  and  fluorides 
are  poisons  because  they  combine  the  cell  calcium,  and  barium  salts 
may  be  poisonous  because  they  precipitate  the  SO4  ions.  We  can 
readily  imagine  that  the  combining  of  even  one  of  the  essential  con- 
stituents of  the  cell  may  so  upset  the  normal  chemical  processes  that 
the  cell  no  longer  takes  up  substances  to  repair  its  waste,  and  hence 
necrosis  ensues. ^^ 

Physical  agents  may  cause  necrosis,  usually  in  ways  too  obvious 
to  require  explanation.  With  most  cells,  large  portions  of  the  cyto- 
plasm can  be  destroyed  without  serious  results,  for  so  long  as  the 
nucleus  is  intact  the  cytoplasm  can  be  reconstructed.  The  fact  that 
necrosis  frequently  follows  relatively  slight  injuries  of  the  nucleus 
is  perhaps  best  explained  by  considering  that  injury  to  the  nuclear 
membrane  modifies  the  permeability  of  the  nucleus  for  substances  in 
solution,  which  might  readily  affect  its  metabolic  activities  to  a  serious 
degree.  It  is  possible,  also,  that  solvents  of  lipoids,  such  as  chloro- 
form, etc.,  produce  much  of  their  deleterious  effects  by  modifying 
the  permeability  of  the  cell,  if  the  semipermeability  of  cell  mem- 
branes depends  largely  upon  the  lipoids  they  contain. ^^ 

Physical  injury  of  even  slight  degree  may  bring  on  severe  alterations 
in  cells,  however,  and  indeed  may  cause  severe  chemical  alterations. 
We  know  that  many  chemical  reactions  can  be  brought  about  by  slight 
mechanical  disturbances,  e.  g.,  the  explosion  of  fulminate,  nitrogen 
iodide,  etc.,  and  it  is  quite  possible  that  mechanical  disturbances  can, 
likewise,  cause  chemical  changes  in  the  protoplasm.  JNIechanical 
injury  of  cells  under  the  microscope  results  in  an  apparent  increase 

52  Jour.  Infect.  Dis.,  1911  (9),  117. 

'3  Amer.  Jour.  Physiol.,  1901  (10),  290;  NichoU,  Jour.  Biol.  Chem.,  1909  (5), 
453. 

^*  It  is  hardly  profitable  here  to  go  further  into  the  theories  of  the  action  of 
poisons,  which  are  generally  extensively  considered  in  the  treatises  on  toxicology 
and  pharmacology  (also  by  Davenport,  loc.  cit). 

5^  See  Pascucci,  Hofmeister's  Beitrage,  1905  (6),  552. 


382  RETROGRESSIVE  CHANGES 

in  the  acid  reaction  of  the  part  involved  (Chambers)  ^^  and  Hkewise 
traumatized  nervous  tissues  develop  an  acid  reaction  (Moore). ^^ 
Many  lower  animals  devoid  of  a  nervous  system  respond  to  mechanical 
stimuli  by  chemical  activity;  e.  g.,  the  production  of  phosphorescence 
by  marine  organisms  when  agitated  by  an  oar,  etc.  Possibly,  the 
secretion  of  thrombokinase  by  the  leucocytes,  which  occurs  whenever 
they  come  in  contact  with  a  foreign  body,  is  an  example  of  a  similar 
reaction  to  a  mechanical  stimulus.  Even  in  urticaria  factitia  the  sim- 
ple mechanical  irritation  which  suffices  to  produce  the  wheals  is  fol- 
lowed very  quickly  by  extensive  nuclear  fragmentation,^^  but  it  may 
be  that  unknown  poisons  are  present  in  the  hypersensitive  skin  and 
cause  the  karyorrhexis,  and  not  the  trauma  alone.  We  have  no  good 
evidence  that  mere  contact  with  a  chemically  inert  foreign  body  unac- 
companied by  cellular  injury,  can  cause  death  of  tissue-cells.^^  How- 
ever, Chambers ^^"  states  that  simple  trauma,  even  mere  compression, 
of  the  eggs  of  asteria  may  cause  them  to  coagulate  into  a  solid  mass. 
Extreme  changes  in  osmotic  pressure  may  lead  to  cell  death,  either 
by  causing  structural  alteration  in  the  cell  (e.  g.,  the  bursting  of  plant- 
cells  in  water),  or  concentration  of  the  electrolytes  may  become  so 
great  that  the  colloids  are  thrown  out  of  solution,  as  in  the  ordinary 
salting-out  processes  of  the  laboratory.  It  is  doubtful,  however,  if 
osmotic  changes  per  se  ever  become  so  abnormal  within  the  animal 
body  (except  in  experimental  conditions)  as  of  themselves  to  cause  cell 
necrosis. 

Varieties  of  Necrosis 

Coagulation  Necrosis.' — This  name  is  applied  to  necrotic  areas 
that  are  firm,  dry,  usually  pale  yellowish  in  color,  and  observed  prin- 
cipally in  areas  of  total  anemia  or  tuberculosis.  The  question  has 
been  long  disputed  as  to  whether  a  true  coagulation  occurs  in  such 
tissues  or  not.  Necrosis  produced  by  heat,  carbolic  acid,  corrosive 
sublimate,  etc.,  is  naturally  a  coagulation  necrosis,  the  cells  of  the 
affected  area  having  undergone  true  coagulation;  i.  e.,  the  conversion 
of  their  soluble  colloids  (sols)  into  the  insoluble  "pedous"  modification. 
Whether  the  same  change  occurs  in  areas  of  anemic  necrosis  is  not  so 
well  established.  If  the  part  contains  a  fair  amount  of  plasma  the 
liberation  of  the  tissue  coagulins  from  the  dead  cells  will  cause  a  con- 
version of  the  fibrinogen  into  fibrin — this  can  usually  be  demonstrated 
microscopically,  but  the  presence  of  fibrin  is  not  constant,  and  its 
quantity  is  usually  insufficient  to  explain  satisfactorily  the  condition 

»«  Amer.  Jour.  Physiol.,  1917  (43),  1. 
"  Proc.  Soc.  Exp.  Biol.  Med.,  1917  (15),  18. 
08  Gilchrist,  Bull.  Johns  Hopkins  Hosp.,  1908  (19),  49. 

90  Meltzer  (Zeit.  f.   Biol.,   1894   (30),  4G4)  has  shown  that  bacteria    may  be 
killed  by  violent  agitation,  which  causes  disintegration  of  the  cells. 
09"  Trans.  Roy.  Soc.  Canada,  1918,  p.  41. 
^  Literature  by  Jores,  Ergebnisse  der  Pathol.,  1898  (5),  IG. 


COAGULATION  NECROSIS  383 

of  coagulation  necrosis  in  infarcts,  etc.,  as  Weifrert  maintained.^ 
Schmaus  and  Albrecht  believe  that  a  true  coagulation  of  the  cell 
proteins  does  occur  in  anemic  infarcts,  etc.,  for  they  found  that  the 
cells  of  kidneys  with  lijrated  vessels  contain  at  first  granules  soluble 
in  water  and  salt  solution;  after  forty-eight  hours  the  granules  cannot 
be  dissolved  in  these  solvents  or  in  weak  acetic  acid,  but  are  soluble 
in  2  per  cent.  KOH;  after  five  to  six  days  the  granules  are  insoluble 
even  in  KOH.  Beyond  these  experiments,  we  seem  to  have  no  proof 
of  the  occurrence  of  intracellular  coagulation  within  areas  of  coagula- 
tion necrosis  due  to  anemia;  exact  chemical  studies  on  this  point  are 
much  needed.  Since  tissue-cells  contain  coagulins  for  fibrinogen,  it  is 
possible  that  they  also  contain  coagulins  for  cell-proteins,  but  this 
remains  to  be  established.  We  do  not  know  whether  Chambers' 
observations  on  the  spontaneous  coagulation  of  tiaumatized  asteria 
eggs^^o  are  applicable  to  other  cells.  Bacteria  produce  substances 
coagulating  milk  and  fibrinogen.  Bergey^  calls  attention  to  the 
coagulation  of  serum  by  enzymes  and  acids  produced  by  bacteria, 
and  RuppeP  found  that  the  tubercle  bacillus  produces  substances 
precipitating  proteins;  hence  coagulation  necrosis  in  bacterial  infec- 
tions may  be  brought  about  in  this  way,  and  SchmolP  has  shown 
that  the  necrosis  occurring  in  tubercles  is  associated  with  an  almost 
complete  coagulation  of  the  cell-proteins. 

Necrosis  associated  with  inflammatory  exudation  is,  of  course,  ac- 
companied by  coagulation  of  the  fibrinogen  of  the  exudate  (e.  g., 
diphtheria);  this  type  of  coagulation  necrosis  is  chemically  a  simple 
fibrin-formation  and  readily  understood.  The  peculiar  hyaline  de- 
generations of  parenchymatous  cells  (e.  g.,  Zenker's  degeneration 
of  muscles)  are  often  included  under  this  class,  but  it  would  seem 
more  probable  that  the  processes  consist  rather  of  the  fusion  of  the 
structural  elements  of  the  cell  into  a  homogeneous  substance  than  a 
true  coagulation.  When  necrosis  is  produced  by  chemical  means 
more  or  less  coagulation  of  some  of  the  soluble  proteins  probably 
takes  place;  even  in  plant  cells  this  coagulation  of  dead  protoplasm 
is  described.^ 

Liquefaction  necrosis  occurs  particularly  in  the  central  nervous 
system,  where  the  cell  substance  seems  not  to  undergo  the  coagulative 

^  Weigert  believed  that  the  dead  area  becomes  permeated  by  plasma  containing 
fibrinogen,  which  is  coagulated  in  and  between  the  cells.  He  put  much  weight 
on  an  increase  in  size  of  the  necrotic  area,  which  is  by  no  means  constant,  as  he 
intimated;  necrotic  areas  are  inelastic,  and  when  death  occurs  thej-  do  not  shrink 
with  the  fall  of  blood  pressure  as  the  surrounding  tissues  do,  and  hence  they 
may  appear  to  project  from  the  surface  of  the  dead  organ  when  thej-  did  not  do 
so  during  life.  According  to  Moos  (Virchow's  Archiv.,  1909  (195),  273)  the  plasma 
does  not  permeate  infarcted  areas  to  the  extent  that  Weigert  assumed. 

3  Jour.  Amer.  Med.  Assoc,  1907  (49),  680. 

*  Zeit.  phvsiol.  Chem.,  1898  (26),  218. 

6  Deut.  Arch.  klin.  Med.,  1904  (81),  163. 

^  Gaidukov.  Zeit.  chem.  KoUoide,  1910  (6),  260;  Lepeschkin,  Ber.  Deut.  Bot. 
Gesell.,  1912  (30),  528. 


384  RETROGRESSIVE  CHANGES 

changes  described  in  the  preceding  paragraphs.  Whether  this  is 
due  to  a  lack  of  tissue-coaguhns  or  to  a  difference  in  cell  composition 
cannot  be  said,  but  the  large  proportion  of  lipoids  in  brain  tissue  is 
probably  an  important  factor.  Probably  "edema  ex  vacuo"  is  re- 
sponsible for  much  of  the  accumulation  of  fluid,  due  to  the  anatomical 
conditions  that  prevent  a  shrinking  or  collapse  of  the  tissues  to  fill 
in  the  gap,  and  the  lack  of  connective-tissue  formation.  Aseptic 
softening  in  general  may  be  safely  ascribed  to  digestion  of  proteins 
by  cellular  enzjnnes,  either  from  the  dead  cells  or  from  the  leucoc3'tes. 
Suppuration  is  merely  a  form  of  liquef active  necrosis,  in  which  such 
digestion  is  particularly  rapid  because  of  the  large  number  of  leucocytes 
that  are  present.  Necrosis  of  the  gastric  mucosa  or  of  the  pancreas  is 
also  followed  by  rapid  liquefaction,  through  the  action  of  the  digestive 
enzymes  of  these  tissues.  When  necrosis  is  accompanied  by  edema 
(as  in  superficial  burns),  the  fluid  enters  the  cells  in  large  amounts, 
and  in  this  way  another  form  of  liquefaction  necrosis  may  be  produced. 
Bacterial  enzymes  may  be  a  factor  in  producing  liquefaction  of  dead 
tissues,  but  with  most  pathogenic  forms  there  is  little  proteolytic 
activity.^ 

Caseation. — This  term  is  applied  to  a  form  of  coagulation  necrosis 
in  which  the  dead  tissue  has  an  appearance  quite  similar  to  that  of 
cheese.  If  we  bear  in  mind  the  fact  that  cheese  is  a  mixture  of  coagu- 
lated protein  and  finely  divided  fat,  and  that  in  caseation  we  have  a 
coagulation  of  tissue  proteins  associated  with  the  deposition  of  con- 
siderable quantities  of  fat,  the  reason  for  the  gross  resemblance  of 
the  product  of  this  form  of  necrosis  to  cheese  is  apparent.  SchmolP 
has  analyzed  caseous  material,  and  found  it  almost  entirel}"  free  from 
soluble  proteins  or  proteoses.  The  protein  material  is  almost  solely 
coagulated  protein,  which  in  its  elementary  composition  is  related  to 
the  simple  proteins  or  to  fibrin,  and  not  at  all  to  the  nucleoproteins. 
The  extremely  small  amount  of  phosphorus  present  in  the  caseous 
material  indicates  that  the  products  of  disintegration  of  the  cell  nuclei 
must  diffuse  out  early  in  the  process.  Caseation  is,  therefore,  char- 
acterized by  a  coagulation  of  the  proteins  and  a  dissolving  outTof  Jhie 
nuclear  components.  Schmoll  does  not  explain  the  cause  of  coagula- 
tion, however.  It  may  be  that  it  is  the  same  as  in  the  coagulation  of 
anemic  infarcts  (since  tuberculous  areas  are  decidedly  anemic),  or 
possibly  the  tubercle  bacillus  produces  substances  coagulating  pro- 
teins, as  Ruppel  states  is  the  property  of  "tuborculosamin."  Indeed; 
Auclair^  claims  that  the  fatty  substance  that  can  be  extracted  from 
tubercle  bacilli  by  chloroform  is  the  cause  of  the  caseation.  Dead 
tubercle  bacilli  do  not  produce  true  caseation,  however,  according  to 
Kelber;^"  hence  the  substance  causing  the  necrosis  evidently  docs  not 

^  See  Bittrolff,  Beitr.  path.  Anat.,  1915  (60),  337. 

8  Deut.  Arch.  Idin.  Med.,  1904  (81),  163. 

9  Arch.  m6d.  exper.,  1899,  p.  363. 

10  Quoted  by  Uurck  and  Oheindorfer,  Ergebnisse  der  Pathol.,  1899  (6),  288. 


CASEATION  385 

diffuse  readily  from  the  bodies  of  the  bacilli.  Comparison  of  the 
chemical  composition  of  bovine  and  human  tuberculous  lesions  witli 
the  corresponding  normal  tissues  by  Caldwell'^  gave  the  following 
results: 

The  tubercle  walls  and  the  caseous  material  from  lymph  gland  tubercles  contain 
a  lower  percentage  of  water  than  does  the  normal  tissue.  In  normal  bovine  liver 
tissue,  the  percentage  of  water  present  is  less  than  that  of  the  tubercle  walls  or  of 
the  caseous  material  from  liver  tubercles.  The  specimens  of  oasQous  material 
from  lymph  gland  and  liver  tubercles  approach  each  other  closely  in  their  water 
content,  the  average  being  about  75%  for  the  bovine  material. 

The  alcohol-ether-soluble  substances  from  normal  bovine  lymph  glands  form 
about  24.4%  of  the  dry  weight,  or  about  4.4%  of  the  moist  weight.  The  walls  of 
the  lymph  gland  tubercles  contain  a  distinctly  larger  amount  of  lipins  than  does 
the  caseous  material  or  the  normal  tissue.  On  the  contrary,  the  walls  of  liver 
tubercles  are  poor  in  lipins  as  compared  with  the  normal  tissue,  and  they  contain 
a  smaller  amount  of  fats  than  does  the  caseous  material  from  these  tubercles. 
When  calculated  on  the  basis  of  the  dry  weight,  the  caseous  material  from  lymph 
gland  tubercles  contains  a  smaller  percentage  of  lipins  than  does  normal  lymph 
gland  tissue.  When  the  ash  is  deducted,  this  difference  disappears  and  the 
content  of  lipins  becomes  equal  to  or  slightly  greater  than  that  of  the  normal 
tissue,  but  less  than  that  of  the  tubercle  walls.  When  calculated  on  an  ash-free 
basis,  the  lipin  content  of  the  caseous  material  from  liver  tubercles  is  distinctly 
less  than  that  of  the  normal  tissue  but  greater  than  the  lipin  content  of  the  tubercle 
walls. 

Cholesterol  forms  about  6.5%  of  the  lipins  from  normal  bovine  lymph  glands, 
or  about  1.5%  of  the  dry  weight.  The  lipins  from  the  walls  of  lymph  gland  and 
liver  tubercles  contain,  in  every  case,  2-3  times  as  much  cholesterol  as  do  the  lipins 
from  the  normal  tissues.  This  is  an  actual  increase  also  when  calculated  on  the 
basis  of  the  dry  weight.  The  caseous  material  contains  even  a  larger  percentage 
of  cholesterol  than  do  the  tubercle  walls.  Phospholipins  constitute  about  32% 
of  the  lipin  fraction  of  normal  bovine  lymph  glands,  or  about  7.9%  of  the  dry  weight; 
the  corresponding  values  for  normal  liver  are  41.2%  of  the  fats,  or  14%  of  the  dry 
weight.  The  phospholipin  content  of  the  fats  from  the  tubercle  walls  is  slightly 
less  than  that  of  the  normal  tissues,  while  there  is  a  very  marked  reduction  in  the 
phospholipin  content  of  the  lipins  from  caseous  material  of  bovine  origin.  In 
the  specimen  of  caseous  material  from  human  lymph  glands,  phospholipins  formed 
30.9%  of  the  total  lipins.  The  iodin  numbers  obtained  from  the  fats  of  the  tuber- 
culous specimens  from  lymph  glands  are  higher  than  those  from  the  normal  tissues. 
This  observation  does  not  hold  true  for  the  liver  specimens.  In  the  latter,  there  is 
no  difference  noted  between  the  iodin  numbers  obtained  for  the  lipins  from  normal 
and  tuberculous  specimens,  although  the  values  are  practically  the  same  as  those 
from  the  fats  from  the  lymph  gland  tubercles. 

In  the  residues  of  caseous  material  left  after  extraction  with  alcohol  and  ether 
the  nitrogen  content  remains  relatively  high;  in  fact,  the  reduction  in  nitrogen 
content  is  only  slight  when  the  calculations  are  made  on  ash-free  residues.  The 
percentage  of  nitrogen  does  not  differ  much  from  that  obtained  from  the  normal 
proteins  of  these  tissues.  In  specimens  of  caseous  material  in  which  there  are  no 
macroscopic  evidences  of  calcification  other  than  the  presence  of  sandlike  particles, 
calcium  sometimes  forms  as  much  as  15%  of  the  residue  left  after  extraction  of  the 
fats.     In  such  residues,  the  phosphorus  content  may  reach  9%. 

The  amount  of  purine  nitrogen  in  the  walls  of  lymph  gland  tubercles  is  only 
slightly  more  than  iiali  that  of  normal  lymph  gland  tissue,  and  the  amount  is 
apparently  much  less  in  the  caseous  material.  In  the  residues  from  the  walls  of 
liver  tubercles,  purine  nitrogen  is  present  in  only  slightly  higher  percentage  than  in 
the  normal  liver.  The  results  here  obtained  would  seem  to  indicate  that  the  pur- 
ines are  even  more  abundant  in  the  caseous  residues  of  liver  tubercles.  The 
amount  of  material  which  enters  the  water  solution  during  extraction  is  distinctly 
less  from  caseous  material  than  from  the  residues  of  normal  tissues. 

"  Jour.  Infect.  Dis.,  1919  (24),  81.  Full  review  on  composition  of  tuberculous 
tissues. 

25 


386  RETROGRESSIVE  CHANGES 

The  abundance  of  fat  in  caseous  material  on  microscopic  exami- 
nation is  very  striking.  In  addition  to  the  figures  obtained  by  Caldwell, 
Bossart^^  found  from  13.7  per  cent,  to  19.4  per  cent,  of  the  dry  sub- 
stance of  caseous  material  soluble  in  alcohol  and  ether.  In  the  scrap- 
ings from  tuberculous  bovine  glands  I  have  found  22.7-23.9  per  cent, 
of  the  organic  material  soluble  in  alcohol  and  ether. ^^  Of  this  soluble 
material,  Bossart  found  25  to  33  per  cent,  of  cholesterol,  and  Leber^'* 
found  38.31  per  cent.,  which  is  a  much  higher  phospholipin  pro- 
portion than  Bossart  detected.  Caldwell  found  cholesterol  higher 
and  phospholipins  lower  in  caseous  than  in  normal  tissues.  The 
total  amount  of  lipins,  however,  constituted  a  smaller  percentage  of 
the  dry  weight  than  in  the  normal  tissues  from  which  the  caseous 
material  originated.  Presumably  these  fatty  materials  are  derived 
chiefly  from  the  disintegrated  cells;  this  is  probably  true  of  the  phos- 
pholipin and  cholesterol,  but  the  fact  that  in  histological  preparations 
most  of  the  fat  is  found  about  the  periphery  of  the  caseous  area,^^  sup- 
ports the  belief  that  it  has  wandered  in  from  the  outside.^®  A  certain 
proportion  of  the  fat  is  possibly  derived  from  the  bodies  of  the  tubercle 
bacilli,  which  usually  contain  about  40  per  cent,  of  fatty  matter;  but 
it  has  not  been  determined  whether  the  fat  from  this  origin  forms  an 
appreciable  part  of  the  fatty  matter  of  caseous  material. 

Caseous  areas  persist  for  extremely  long  periods  of  time  without 
undergoing  absorption,  which  indicates  that  the  autolytic  enzymes 
are  destroyed  early  in  the  process,  presumably  by  the  toxins  of  the 
tubercle  bacillus;  corresponding  to  this  Schmoll  found  autolj'sis  very 
slight  indeed  in  caseous  areas,  and  even  when  the  caseous  material 
breaks  down  to  form  a  "cold  abscess"  the  fluid  differs  from  true  pus 
in  containing  less  free  amino-acids,  e.  g.,  tyrosine  is  missing."  Caldwell 
also  obtained  lower  figures  for  extractives  in  caseous  than  in  normal 
tissues.  Because  of  a  lack  of  chemotactic  substances  no  leucoc3'tes 
enter  to  remove  the  dead  material,  in  consequence  of  which  caseous 
material  gives  no  evidence  of  containing  proteases,  according  to  the 
Miiller-Jochmann  plate  method.  That  the  failure  of  absoprtion  is  not 
due  to  a  modification  of  the  proteins  into  an  indigestible  form  is 
shown  by  the  rapid  softening  of  caseous  areas  when,  through  mixed 
infection,  chemotactic  substances  are  once  developed  and  leucocytes 
enter.  Jobling  and  Petersen^^  suggest  that  in  caseation  the  autolysis 
is  inhibited  by  the  soaps  of  fatty  acids,  which  are  abundant  in  caseous 
areas  and  have  a  marked  antitryptic  effect. 

^*  Quoted  by  Schmoll,  loc.  cit.^ 
"  Wells,  Jour.  Med.  Research,  1906  (14),  491. 
^*  Quoted  by  Schmoll.^ 
1^  Sata,  Ziegler's  Beitr.,  1900  (28),  461. 

^^  Fischler  and  Gross  (Ziegler's  Beitr.,  1905  (7th  suppl.),  344)  could  find  no 
fatty  acids  in  caseous  areas  bv  histological  methods, 
i'  See  Muller,  Cent.  inn.  Med.,  1907  (2S),  297. 
IS  Jour.  Exp.  Med.,  1914  (19),  239;  Zcit.  Immunitat.,  1914  (23),  71. 


PANCREATIC  FAT  NECROSIS  387 


Fat  Necrosis" 


ThrouKh  iisaj.'c  (his  Icrm  Ikis  come  to  indicate  a  specific  form  of 
necrosis  of  fat  tissue,  which  is  characterized  l)y  a  focal,  circumscribed 
arran<i,ement,  and  by  the  splitting  of  the  fat  in  the  necrotic  area  into 
fatty  acids  and  glycerol,  the  latter  disappearing,  the  former  com- 
bining with  bases  to  form  soaps. ^^  In  practically  all  cases  fat  necrosis 
is  produced  by  the  action  of  pancreatic  juice  upon  fat  tissue,*^'  pre- 
sumably through  the  action  of  the  enzymes  it  contains,  and  the  con- 
dition can  be  produced  experimentally  b}'-  any  procedure  that  causes 
escape  of  the  pancreatic  juice  from  ito  natural  channels. 

Langerhans"  made  the  first  studies  cf  the  nature  of  the  changes 
in  fat  necrosis  and  established  the  fact  that  the  fat  of  the  cells  is 
split  into  its  components,  and  that  the  fatty  acids  combine  (at  least  in 
part)  with  calcium.  Dettmer-^  found  that,  although  fresh  pancreatic 
juice  caused  fat  necrosis,  a  commercial  preparation  of  trypsin  did  not 
do  so,  and,  therefore,  he  concluded  that  probably  the  lipase  of  the 
pancreatic  juice  was  the  active  agent.  Flexner-"*  supported  this  con- 
tention by  demonstrating  the  presence  of  a  fat-splitting  enzyme  in 
foci  of  fat  necrosis,  which  was  corroborated  by  Opie.^-^  The  latter^® 
was  also  able  to  demonstrate  the  presence  of  Kpase  in  the  urine  of  a 
patient  with  fat  necrosis,"  and  the  highest  values  for  amylase  in  the 
blood  and  urine  are  found  in  pancreatitis  (Stocks).-^ 

In  a  study  of  the  pathogenesis  of  fat  necrosis,  particularly  with 
reference  to  the  question  whether  the  lipase  or  the  trypsin  of  the 
pancreatic  juice  was  responsible,  Wells-^  found  that  typical  fat  necro- 
sis could  be  produced  by  injecting  extracts  of  fresh  pancreas  into 

^^  General  literature  will  be  found  in  the  articles  cited  in  the  text;  also  in 
Opie's  "Diseases  of  the  Pancreas;"  and  in  Truhart's  "Pankreas-Patholoeie," 
Wiesbaden,  1902. 

-"  The  fatty  acids  form  masses  of  crystals  in  the  fat-cells,  and  they  can  also 
be  demonstrated  microchemically  by  Benda's  method  (Virchow's  Arch.,  1900  (161), 
194),  which  consists  of  staining  with  a  copper  acetate  mixture,  blue-green  copper 
salts  of  the  fatty  acids  being  formed. 

21  Wulff  (Berl.  klin.  Woch.,  1902  (39),  734),  claims  to  have  observed  an  excep- 
tion to  this  rule,  but  his  account  is  not  by  itself  convincing.  Fabyan  (Johns 
Hopkins  Hosp.  Bull.,  1907  (18),  349)  reports  a  case  of  multiple  subcutaneous 
fat  necrosis  without  pancreatic  lesions,  in  a  14  days'  old  baby,  and  gives  a  re- 
view of  other  similar  cases.  This  case,  however,  may  be  one  of  scleroderma 
C.  S.  Smith,  Jour.  Cut.  Dis.,  1918  (36),  436). 

"  Virchow's  Arch.,  1890  (122),  252. 

^'Dissertation,  (Jottingen,  1S95. 

24  Jour.  Exper.  Med.,  1897  (2),  413. 

"  Contrib.  of  pupils  of  W.  H.  Welch,  Baltimore,  1900,  p.  859;  Johns  Hopkins 
Hosp.  Rep.,  1900  (9),  859. 

-^  Opie,  "Diseases  of  the  Pancreas,"  Lippincott,  1903,  p.  156;  Johns  Hopkins 
Hosp.  Bull.,  1902  (13),  117. 

2^  It  yet  remains  to  be  seen  if  this  is  a  constant  occurrence,  and  also  if  the 
lipase  so  excreted  comes  from  the  pancreas,  for  Zeri  (11  Policlinico,  1905  (12),  733) 
has  found  lipase  in  the  urine  in  hemorrhagic  nephritis  and  inflammation  of  the 
urinary  tract;  also  Pribram  and  Loewv,  Zeit.  physiol.  Chem.,  1912  (76),  489. 

28  (^lart.  Jour.  Med.,  1916  (9),  216.' 

"  Jour.  Med.  Research,  1903  (9),  70. 


388  RETROGRESSIVE  CHANGES 

animals,  either  of  the  same  species  as  that  from  which  the  pancreas 
was  obtained,  or  into  a  foreign  species.  Commercial  "pancreatins" 
were  also  quite  effective,  whether  in  weak  acetic  acid  or  weak  alkaline 
solutions.  The  power  of  these  materials  to  cause  fat  necrosis  was 
reduced  by  heating  to  or  above  60°  for  five  minutes,  and  completely 
destroyed  at  71°,  indicating  that  the  active  agent  is  an  enzyme.  But, 
as  in  the  same  material  trypsin  was  injured  by  temperatures  above  60°, 
and  destroyed  at  between  70°  and  72°,  and  lipase  was  weakened  above 
50°,  and  destroyed  above  70°,  it  was  impossible  to  determine,  by 
heating  pancreatic  preparations,  whether  the  lipase  or  the  trypsin 
was  the  essential  factor.  By  permitting  pancreatic  extracts  to  digest 
themselves  it  was  found  that  the  power  to  produce  fat  necrosis 
decreased,  pari  passu,  with  the  decrease  in  lipolytic  strength.  Prepara- 
tions strongly  tryptic,  but  very  weak  in  lipase,  produced  no  fat  necro- 
sis, and,  on  the  other  hand,  extracts  of  pig's  liver  or  of  cat's  serum, 
both  rich  in  lipase  but  devoid  of  trypsin,  were  equally  ineffective. 
Furthermore,  mixtures  of  hver  or  serum  lipase  and  trypsin  were 
incapable  of  causing  fat  necrosis.  Fresh  pancreatic  extracts  from 
fasting  dogs,  containing  lipase  but  almost  no  trypsin  (which  in  fresh 
extracts  is  still  in  the  form  of  inactive  trypsinogen),  produced  abun- 
dant fat  necrosis,  whereas  after  the  trypsinogen  in  such  extracts  was 
activated  by  enterokinase,  no  fat  necrosis  could  be  produced.  It 
therefore  seems  certain  that  trypsin  alone  cannot  produce  fat  necrosis, 
and  that  the  decrease  in  strength  of  lipase  in  a  pancreatic  extract  is 
associated  with  a  corresponding  decrease  in  power  to  produce  fat 
necrosis.  But,  on  the  other  hand,  lipase  of  liver  or  blood-serum  alone, 
or  when  mixed  with  trypsin,  will  not  produce  fat  necrosis.  The  possi- 
bility remains  that  pancreatic  lipase  is  different  from  liver  or  serum 
lipase,  and  can  by  itself  cause  fat  necrosis;  more  probably,  however, 
the  production  of  fat  necrosis  depends  upon  a  double  action,  trypsin 
causing  the  death  of  the  cells,  and  lipase  splitting  the  fat?.^"  The 
fatty  acids  alone  will  not  cause  necrosis  of  fat-cells,  and  it  was  shown 
that  the  first  steps  in  the  process  consist  of  a  necrosis  of  the  surface 
endothelium  extending  into  the  connective  and  fat  tissue;  this  may 
occur  in  a  few  minutes,  while  evidence  of  fat-splitting  can  be  obtained 
only  after  about  three  hours,  and  the  splitting  occurs  only  in  cells 
that  have  already  become  necrotic;  hence  the  fat-splitting  is  not  the 
cause  of  the  necrosis,  but  occurs  subsequent  to  the  necrosis.     After 

^°  When  fat  tissue  dies  in  the  body  from  other  causes,  the  lipase  normally  con- 
tained within  the  fat  tissue  does  not  cause  the  changes  seen  in  fat  necrosis.  It  is 
possible,  therefore,  that  the  combining  of  newly  split  fatty  acids  by  the  alkali  of 
the  pancreatic  juice  is  responsible  for  the  formation  of  the  large  amount  of  soaps 
found  in  fat  necrosis.  Otherwise  we  might  expect  the  lipase  to  produce  only 
an  equilibrium,  and  that,  in  the  case  of  fat,  seems  to  exist  when  most  of  the  sub- 
stance is  neutral  fat.  In  support  of  this  idea  1  found  that  strong  alkalies  injected 
into  fat  tissue  sometimes  caused  changes  very  closely  resembling  areas  of  fat 
necrosis  in  the  early  stages. 


PANCREATIC  FAT  NECROSIS  389 

about  four  hours  a  substance  appears  in  the  decomposed  fat  that 
stains  with  hematoxylin,  which  is  probably  calcium. 

Fat  necrosis  may  be  produced  by  any  means  that  will  cause  the 
escape  of  pancreatic  juice  from  the  natural  channels  within  the 
gland.  In  human  pathology  it  has  followed  trauma  and  acute  in- 
fection of  the  gland,  and  the  blocking  of  the  ampulla  of  Vater  by  gall- 
stones which  permits  the  bile  to  back  up  into  the  pancreatic  duct, 
where  it  produces  an  acute  inflammation  of  the  pancreas  (Opie).^^ 
Flexner^^  has  shown  that  it  is  the  bile  salts  that  cause  the  inflamma- 
tion, and  also  that  this  effect  is  decreased  or  prevented  by  the  presence 
of  large  amounts  of  colloids.  Much  emphasis  is  laid  by  some  au- 
thors^^  upon  the  necessity  of  enterokinase  passing  up  the  ducts  to 
activate  the  trypsinogen  (an  idea  first  advanced  by  Starling  and  Bay- 
liss  in  1902),  but  it  should  be  remembered  that  there  are  kinases  pres- 
ent in  leucocytes,  and  that  kinases  can  develop  in  the  pancreas  itself 
during  autolysis,  which  can  activate  the  trypsinogen;  hence  the  pres- 
ence of  e nter o-kinase  is  not  essential  for  sufficient  activation  of  tryp- 
sinogen to  account  for  pancreatitis  and  fat  necrosis.  Lattes^*  believes 
that  fresh  pancreatic  juice,  which  digests  tissues  very  slowly,  can  pro- 
duce typical  fat  necrosis  but  not  the  characteristic  intoxication;  this 
results  from  the  action  of  juice  which  has  been  activated  by  entero- 
kinase, or  by  products  of  pancreatic  autolysis  which  have  a  similar 
effect.  The  kinases  of  leucocytes  he  found  unable  to  activate  pan- 
creatic trypsinogen  sufficiently  to  make  it  highly  toxic.  These  ob- 
servations indicate  that  in  pancreatic  necrosis  it  is  the  kinase  liberated 
from  the  autolyzing  necrotic  tissue  which  is  responsible  for  the  acti- 
vation and  resulting  toxic  effects  of  the  trypsinogen.  As  a  result  of 
injury  by  bile  salts,  or  any  other  agent  that  produces  cell  death,  the 
dead  and  injured  cells  are  digested  by  the  pancreatic  juice  which  is 
thus  further  activated  and  makes  its  escape  into  the  surrounding  fat 
tissue.  Wells'  experiments  showed  that  the  lesions  of  fat  necrosis 
may  be  produced  in  three  to  five  hours,  large  enough  to  be  visible  to 
the  naked  eye;  their  form  and  size  depend  solely  upon  the  area  of  fat 
tissue  exposed  to  the  action  of  the  pancreatic  juice.  The  process 
progresses  for  but  a  few  hours,  the  extension  seeming  to  be  limited  by 
surrounding  leucocytes.  The  lesions  may  appear  at  remote  points  in 
the  thoracic  and  pericardial  cavities  or  in  the  subcutaneous  tissues, 
the  causative  agent  probably  being  carried  by  the  lymphatic  vessels, 
possibly  in  the  form  of  emboli  of  pancreas  cells. ^^  There  may  even  be 
some  splitting  of  the  fats  in  the  liver  in  these  cases,  with  intrahepatic 

"  Bull.  Johns  Hopkins  Hosp.,  1901  (12),  182. 
32  Jour.  Exp.  Med.,  1906  (S),  167. 

33P61ya,  Mitt.  Grenz.  Med.  u.  Chir.,  1911  (24),  1;  Rosenbach,  Arch.  klin. 
Chir.,  1910  (93),  278. 

^*  Virchow's  Arch.,  1913  (211),  1. 

35Pavr  and  Martina,  Deut.  Zeit.  Chir.,  1906(83),  189. 


390  RETROGRESSIVE  CHANGES 

necrosis.  ^^  Fat  necrosis  itself  is  not  dangerous  to  the  affected  organ- 
ism, the  associated  pancreatitis  (and  peritonitis)  catfsing  all  the 
symptoms. ^^  There  is  no  evidence  that  sufficient  quantities  of  soaps 
(which  are  toxic)  are  absorbed  from  the  necrotic  areas  to  cause  appre- 
ciable intoxication.  The  soaps  that  are  formed  in  the  necrotic  areas, 
indeed,  are  probably  not  much  absorbed,  but  are  precipitated  as  cal- 
cium soaps;  in  such  areas  at  least  as  high  as  85  per  cent,  of  the  soaps 
may  be  insoluble. ^^  Healing  follows  rapidly  in  case  of  recovery;  the 
foci  may  disappear  as  early  as  eleven  days  after  their  formation  (in 
experimental  animals). 

In  the  urine  of  persons  with  pancreatitis  is  frequently  found  a 
substance  forming  an  osazone,  which  has  been  the  subject  of  much 
investigation  because  of  its  possible  diagnostic  value.  Cammidge,^^ 
who  first  described  a  reaction  based  on  this  observation,  considers 
that  the  substance  is  a  pentose,'*"  derived  from  the  nucleoproteins  of  the 
pancreas;  it  bears  no  relation  to  the  fat  necrosis,  but  is  commonly 
found  with  fat  necrosis  because  of  the  associated  pancreatitis.  Pre- 
sumably cell  necrosis  elsewhere  than  in  the  pancreas  may  at  times 
cause  the  same  reaction  to  appear.^^  In  pancreatitis  with  fat  necrosis, 
or  whenever  there  is  any  injury  to  the  pancreas,  there  may  be  found 
an  increase  in  the  amount  of  diastase  in  the  blood  and  urine,  sufficient 
to  be  of  diagnostic  value  according  to  Y.  Noguchi.^-  The  peritoneal 
exudate  in  acute  pancreatitis  is  not  toxic,  contains  no  free  trypsin  and 
is  no  more  lipolytic  than  normal  serum,  presumably  because  of  neu- 
tralization of  the  enzymes  and  poisons  by  the  exuded  plasma.*^ 

3«  See  Berner,  Virchow's  Arch.,  1907  (187),  360. 

"  Guleke  (Arch.  klin.  Chir.,  1908  (85),  615)  considers  the  intoxication  of  acuie 
pancreatitis  as  an  intoxication  with  trypsin,  which  can  be  checked  by  antitrypsin. 
Doberauer  (Beitr.  kUn.  Chir.,  1906  (48),  456),  Egdahl  (Jour.  Exp.  Med.,  1907  (9), 
385),  Petersen,  Jobling,  and  Eggstein  {ibid.,  1916  (23),  491),  and  Cooke  and 
Whipple  {ihid.,  1918  (28),  222),  however,  look  upon  the  products  of  cellular  dis- 
integration as  the  source  of  the  intoxication,  v.  Bergmann  (Zeit.  exp.  Path.  u. 
Ther.,  1906  (3),  401),  states  that  the  toxicity  is  not  due  to  either  the  enzymes  or 
to  albumoses;  and  that  it  is  a  true  autointoxication  which  can  be  prevented  by 
previous  immunization  with  either  pancreas  extracts  or  commercial  trypsin.  (See 
also  Fischler,  Deut.  Arch.  klin.  Med.,  1911  (103),  156;  and  v.  Bergmann  and 
Guleke,  Miinch.  med.  Woch.,  1910  (57),  1673.)  The  histones  and  protamines 
liberated  from  the  digested  tissue,  ttnd  which  are  very  toxic,  have  boon  suggested 
as  a  possible  factor  by  Schittenhelm  and  Weichardt  (Zeit.  Immunitiit.,  1913  (14), 
609),  while  the  beta-nucleo-proteins  are  included  among  the  toxic  elements  by 
Goodpasture,  Jour.  Exp.  Med.,  1917  (25),  277. 

^*  See  Frugoni  and  Stradiotti,  Arch.  Sci.  Med.,  Torino,  1910;  also  Berl.  klin. 
Woch.,  1910  (47),  386. 

39  Proc.  Royal  Soc.  Med.,  1910  (III,  pt.  2),  163,  bibliography;  Lancet,  1914, 
Sept.  26. 

"  Weber  believes  that  it  is  a  hexose  (Deut.  med.  Woch.,  1912  (38),  166),  and 
it  may  be  urinary  dextrin  (Pekelharing  and  Van  Koogenhuyze,  Zeit.  physiol. 
Chem.,  1914  (91;,"  151). 

*'  See  Whipple,  cl  al.,  Johns  Hopkins  IIosp.  Bull.,  1910  (21),  339;  Karas,  Zeit. 
klin.  Med.,  1913  (77),  125. 

^2  Arch.  klin.  Chir.,  1912  (98),  11.  2. 

"  Wliipi)le  and  Goodpasture,  Surg.,  Gyn.  and  Obst.,  1913  (17),  541. 


GANGRENE  391 

Self-digestion  of  the  pancreas  occurs  soon  after  death,  and  the  pancreatic 

juice  may  in  ^his  way  bring  about  a  portmortom  fat  digestion  that  rnsornbles 
soiHowhat  tho  intravital  fat  necrosis  in  its  fzjross  appearances/*  and  Wells  found 
that  the  same  changes  might  ne  j)ro(luced  by  injecting  pancreatin  into  the  bodies 
of  dead  animals,  or  by  keeping  fat  tissue  in  pancreatin  solutions.  WuUT  found 
that  fatty  acids  were  demonstrable  by  Benda's  method  in  the  pancreas  of  nearly 
all  cadavers.  The  process  differs  from  the  intra  ntam  form  in  being  le.ss  sharply 
circumscribed,  and  microscopically  by  the  absence  of  cellular  and  vascular  reaction. 
That  the  essential  changes  of  fat  necrosis  can  be  produced  postmortem  is  final 
proof  that  they  arc  due  to  enzymes,  rather  than  to  circulatory  or  cellular  action. 

GANGRENE 

This  term  indicates  merely  that  certain  marked  secondary  changes, 
either  putrefaction  or  desiccation,  have  occurred  in  necrotic  areas  of 
some  size.  Hence  we  have  the  chemical  changes  of  putrefaction 
added  to  those  of  necrosis  in  the  case  of  moist  gangrene,  whereas  in 
dry  gangrene  nearly  all  the  .chemical  changes  are  brought  to  a  stand- 
still through  the  desiccation.  In  the  latter  it  is  only  at  the  line  of 
demarcation,  where  some  moisture  remains,  that  chemical  changes 
still  go  on;  these  consist  chiefly  of  autolysis  of  the  dead  tissues,  and 
also  of  their  digestion  by  leucocytes,  which  results  eventually  in  the 
separation  of  the  dead  tissue  from  the  living;  this  is  best  seen  after 
surface  burns,  carbolic-acid  gangrene,  etc. 

Moist  gangrene  is  accompanied  by  the  dual  action  of  the  cellular 
enzymes  and  of  the  putrefactive  organisms  that  are  growing  in  the 
dead  tissue,  and  as  a  result  such  tissue  contains  all  the  innumerable 
products  of  the  decomposition  of  proteins  and  fats.  Thus  Ziegler 
mentions  as  morphological  elements  that  may  be  present  in  gangren- 
ous tissue:  Fat  needles,  the  so-called  "margarin"  crystals  (a  mixture 
of  stearic  and  palmitic  acids),  fine  acicular  crystals  of  tyrosine,  globules 
of  leucine,  rhombic  plates  of  triple  phosphate,  black  and  brown  masses 
of  pigment,  and  crystals  of  hematoidin.  In  solution  we  also  have, 
beyond  a  doubt,  all  the  substances  formed  in  the  decomposition  of 
proteins,  from  proteoses  and  peptones  down  through  the  different 
amino-acids  to  such  final  products  as  ammonia  and  its  salts,  while  CO2 
and  H2S  are  abundantly  given  off.  In  addition  occur,  undoubtedly, 
many  of  the  ptomains  which  are  formed  by  the  action  of  the  bacteria 
upon  the  amino-acids  derived  from  the  proteins.'*^  In  the  sputum  from 
pulmonary  gangrene  there  is  but  little  soluble  protein,  most  of  the 
nitrogen,  of  which  there  is  much,  is  in  the  formed  elements. ^^  The 
fetid  plugs  which  occur  in  the  bronchioles  in  gangrene,  the  "Dit- 
trich's  plugs,"  were  found  by  Traube  to  be  composed  chiefly  of  fatty 

"  Chiari,  Zeit.  f.  Heilk.,'lS96  (17),  69;  Pforringer,  Virchow's  Arch.,  1899  (158), 
126;  Liepmann,  ibid.,  1902  (169),  532;  WulfT,  Berl.  klin.  Woch.,  1902  (39),   734. 

*^  An  interesting  observation  concerning  gangrene  of  the  lung  has  been  made 
by  Eijkman  (Cent.  f.  Bakt.,  Abt.  1,  1903  (35),  1),  who  found  in  this  condition 
bacteria  that  secrete  an  enzvme  dissolving  elastic  tissue. 

*«Orszag,  Zeit.  klin.  Med.,  1909  (67),  204. 


392  RETROGRESSIVE  CHANGES 

acid  crystals,  and  Schwartz  and  Kayser*^  ascribe  their  formation  to 
the  action  of  lipolytic  staphylococci. 

If  the  necrotic  tissue  is  in  contact  with  living  tissue  over  a  con- 
siderable area,  enough  of  these  products  of  autolysis  and  putrefaction 
may  be  absorbed  to  cause  intoxication  (sapremia).  At  the  same  time, 
the  formation  of  such  large  quantities  of  crystalloids  from  the  pro- 
teins of  the  dead  tissue  leads  to  a  diffusion  of  water  into  this  area, 
with  consequent  swelling,  and  often  a  lifting  up  of  the  skin  in  the 
form  of  blisters. 

Emphysematous  gangrene, ^^  usually  produced  by  gas-forming  an- 
aerobic bacteria,  including  B.  aerogenes  capsulatus,  may  also  possibly 
be  produced  by  B.  coli  communis  in  diabetic  patients  in  whose  blood 
and  tissues  there  may  occur  sufficient  sugar  to  permit  of  gas-formation. 
Hitschmann  and  Lindenthal  found  that  the  gas  produced  in  cultures 
by  an  anaerobic  organism  which  they  isolated  fi'om  a  case  of 
emphysematous  gangrene,  consisted  of  67.55  per  cent,  hydrogen, 
30.62  per  cent,  carbon  dioxide,  and  traces  of  ammonia  and  nitrogen; 
this  corresponds  to  the  statement  of  Welch  and  Nuttall  that  the  gas 
in  the  tissues  of  infected  animals  is  inflammable.  Dunham^^  found 
that  the  gas  produced  by  B.  aerogenes  capsulatus  in  cultures  has  the 
following  composition:  Hydrogen,  64.3  per  cent.;  carbon  dioxide, 
27.6  per  cent.;  other  gases,  probably  chiefly  nitrogen,  8.1  per  cent. 
Grown  in  a  medium  of  muscle  and  water.  Wolf ^°  found  70-75  per  cent 
of  CO2  produced  by  B.  sporogeiies,  while  B.  Welchii  produced  38  per 
cent,  of  CO2,  the  rest  being  chiefly  H.  The  former  bacillus  is  very 
actively  proteolytic,  the  latter  less  so.  Organisms  of  this  group  pro- 
duce much  volatile  organic  acid  which  is  probably  an  important  factor 
in  the  local  necrosis,  especially  in  producing  a  negative  chemotaxis; 
it  may  also  contribute  to  the  acidosis  of  the  disease.  ^^ 

RIGOR  MORTIS 

This  topic  may  be  appropriately  considered  in  connection  with  cell 
death,  since  it  is  a  characteristic  change  occurring  after  general 
death.  All  forms  of  muscle,  striped,  smooth,  and  cardiac,  undergo 
this  change,  which  is  shown  by  a  shortening  and  thickening  of  the 
muscle,  which  also  becomes  opaque  and  hard.  Rigor  mortis  begins 
first  in  the  heart  muscle,  according  to  Fuchs,^^  but  it  is  generally 
observed  first  in  the  eyelids,  then  in  the  muscles  of  the  jaw,  from 
which  point  it  proceeds  downward,  although  the  upper  extremities 

*'  Zeit.  klin.  Med.,  1905  (56),  111. 

"«  Complete  literature  by  Weinberg  and  S^guin,  "La  Gangrene  gazeuse,"  Paris, 
1918. 

"Johns  Hopkins  Hosp.  Bull.,  1897  (8),  68. 

60  Jour.  Path.  Pact.,  1919  (22),  270. 

61  Wrifrht  and  Fleming,  Lancet,  1918  (i),  205. 

62  Literature,  see  v.  Furth,  Ilandhuch  d.  Biochem.,  1909  (II  (2),  252;  also 
Meltzcr  and  Auer,  Jour.  E.xp.  Med.,  190S  (10),  45). 

"Zeit.  f.  Ileilk.,  1900  (21,  Path.  Abt.),  1. 


RIGOR  MORTIS  393 

may  not  become  rigid  before  tlie  lower.  Tlie  time  of  onset  is  ex- 
tremely variable,  but  the  following  general  rules  may  be  stated:  All 
conditions  that  lead  to  excc^ssive  muscular  metabolism,  with  its  re- 
sulting increase  in  the  acidity  of  the  muscle  fluids,  will  hasten  the 
onset  of  rigor  mortis;  thus,  people  killed  suddenly  during  violent 
activitj''  may  remain  almost  in  the  position  in  which  they  met  death. 
Acute  fevers,  strychnine  poisoning,  tetanus,  etc.,  cause  likewise  a 
rapid  onset  of  rigor,  which  may,  indeed,  appear  almost  simultane- 
ously with  death  or  even  before  the  heart  has  stopped  beating. 
When  a  healthy  individual  meets  death  without  previous  exertion, 
rigor  does  not  usually  appear  for  four  or  six  hours,  but  will  be 
hastened  by  heat  and  retarded  by  cold.  Death  from  hemorrhage  or 
asphyxia  is  followed  by  a  slow  development  of  the  rigor.  Under 
ordinary  conditions  rigor  usually  begins  between  the  first  and  second 
hour  after  death  and  is  complete  in  one  or  two  more  hours. ''^ 

The  duration  of  rigor  mortis  also  is  influenced  by  many  factors. 
In  general,  it  may  be  said  that  the  duration  is  in  inverse  relation 
to  the  rapidity  of  onset,  and  directly  to  the  musculature  of  the  in- 
dividual. Therefore,  in  an  emaciated  individual  dying  with  fever, 
rigor  may  appear  and  disappear  again  within  two  or  three  hours,  or, 
indeed,  escape  observation  altogether.  The  body  of  a  muscular  man 
dying  from  accident  or  hemorrhage  may,  on  the  other  hand,  show 
rigor  for  two  or  three  weeks  if  kept  in  a  cold  place.  Once  the  rigor 
has  been  broken  by  force,  it  does  not  again  return. 

Rigor  mortis  may  be  produced  even  before  death,  through  poisons 
(monobromacetic  acid,  quinine),  and  its  occurrence,  even  postmor- 
tem, does  not  necessarily  mean  that  the  muscle  is  dead,  for  if  the  part 
is  transfused  with  a  salt  solution  the  rigor  may  be  removed,  and  the 
muscle  will  then  be  found  to  react  to  stimuli.  This  indicates  that  the 
chemical  changes  of  rigor  mortis  are  not  very  profound. ^^ 

The  chemistry  of  the  changes  involved  in  rigor  mortis  has  been 
a  much-contested  problem.  Two  chief  doctrines  have  been  sup- 
ported: one  that  rigor  was  not  essentially  different  from  ordinary 
muscular  contraction  except  in  degree,  and  perhaps  due  to  a  loss 
of  inhibition  to  contraction.  The  other  looks  upon  it  as  a  coagulation 
similar  to  the  coagulation  of  the  blood;  and  this  idea,  it  may  be  said, 
has  had  the  most  general  acceptance.  Briicke  in  1842  supported 
this  view,  and  in  1859  Kiihne  extracted  from  muscle  a  plasma  which 
coagulated  like  ordinary  blood  plasma.  The  protein  w'hich  formed 
the  clot  is  called  myosin,  and  its  coagulable  antecedent,  myosinogen. 

This  experiment  has  been  since  repeatedly  verified  and  amplified, 
especially  by  v.  Fiirth  and  by  Halliburton,^'^  who  have  separated  more 

**  Rigor  mortis  may  develop  in  the  dead  fetus  while  in  the  womb,  but  it  gen- 
erally disappears  within  five  or  six  hours.  Literature  by  Wolff,  Arch.  f.  Gyn., 
1903  (68),  549;  Das,  Brit.  Jour,  of  Obstet..  1903  (4),  545. 

"  See  Mangold,  Pfltiger's  Arch.,  1903  (96),  498. 

"  "Chemistry  of  Muscle  and  Nerve,"  1904. 


394  RETROGRESSIVE  CHANGES 

definitely  the  proteins  concerned  in  coagulation,  and  found  them 
to  be  globulins.  There  seem  to  be  two:  one,  coagulating  at  47°, 
called  paramyosinogen  (Halliburton),  constitutes  but  about  one-fifth 
of  the  total  clotting  globulin,  and  passes  readily  into  the  insoluble 
clot,  myosin',  the  other,  which  coagulates  at  56°,  constitutes  the  re- 
maining four-fifths  is  called  myosinogen  (Halliburton),  or  myogen 
(v.  Fiirth),  and  before  becoming  changed  into  myosin  it  passes  through 
a  soluble  stage  called  soluble  myogen- fibrin,  which  is  coagulated  at  the 
remarkably  low  temperature  of  40°. 

By  analogy  with  fibrin-formation  we  should  expect  this  clotting 
also  to  be  brought  about  by  an  enzyme,  but  this  has  not  been  proved. 
Calcium  is  of  influence,  favoring  coagulation  greatly,  but  its  presence 
is  not  absolutely  essential  (v.  Fiirth).  Of  particular  importance  is 
the  acid  reaction  of  the  dead  muscle.  Normal  muscle  is  amphoteric 
when  at  rest,  but  when  active  the  reaction  becomes  more  and  more 
acid,  as  it  also  does  when  the  circulation  is  shut  off,  and  hence  acidity 
increases  greatly  after  death.  The  acidity  is  due  chiefly  to  lactic 
acid  (although  the  neutral  phosphates  may  become  converted  into 
acid  phosphates  in  the  presence  of  the  lactic  acid,  and  thus  seem  to 
contribute  to  the  acidity),  and  may  increase  in  twenty-four  hours 
after  death  by  from  6.7  to  12.8  c.c.  of  "/lo  acid  for  each  100  grams 
of  muscle  (v.  Fiirth").  The  same  author  found  that  although  the 
amount  of  acid  might  become  in  time  sufficient  to  cause  coagula- 
tion of  the  muscle  proteins  by  itself,  yet  actually  rigor  mortis  appears 
before  the  acidity  has  reached  any  such  degree.  Verzar^^  saj^s  that  by 
vital  stains  it  can  be  shown  that  in  vital  contraction  no  precipitation 
occurs,  but  it  does  take  place  in  rigor  mortis.  Mcigs^^  advanced 
the  hypothesis  that  the  rigor  is  due  to  the  swelling  of  the  muscle  col- 
loids under  the  influence  of  acids,  a  view  which  is  accepted  by  von 
Fiirth  and  Lenk."^"  When  sufficient  acid  is  formed  in  the  muscle 
the  swelling  may  be  so  great  that  the  structure  of  the  muscle  cell  is 
destroyed  entirely,  and  it  goes  into  the  condition  of  ''waxy  degenera- 
tion."" This  readily  explains  why  the  time  of  appearance  of  rigor 
is  so  modified  by  the  amount  of  muscle  metabolism  before  death.  It 
is,  indeed,  possible  to  produce  rigor  in  living  animals  by  transfusing 
a  limb  with  slightly  acid  salt  solution,'^-  and  in  strychnine-poisoning 
the  nauscular  spasm  may  pass  imperceptibly  into  rigor  mortis. 

"  Hofmeister's  Beitr.,  1903  (3),  543;  see  also  Fletcher  and  Hopkins,  Jour,  of 
Physiol.,  1907  (35),  247;  Wacker,  Biochcm.  Zeit.,  1916  (75),  101. 

"  ]5ioohom.  Zeit.,  1918  (90),  (53. 

"  Amor.  Jour.  Physiol.,  1910  (26),  191. 

"OBiochem.  Zeit.,  1911  (33),  341;  Wien.  klin.  Woch.,  1911  (24),  1079. 

"  Wells,  Jour.  Exper.  Med.,  1909  (11),  1.  Corroborated  ^hv  Stcniinler, 
Virchow's  Arch.,  1914  (216),  57. 

"2  The  hardness  of  a  limb  from  which  the  blood-supply  has  been  shut  off  by 
throml)(jsi.s  or  embolism,  and  also  much  of  the  crami)-like  pain,  is  probably  due 
to  riffor  mortis  in  the  muscles  caused  by  acid  formation  under  conditions  of  sub- 
oxidation. 


ATROPHY  395 

It  has  been  suggested  that  the  chsappcarance  of  rigor  mortis  depends 
upon  beginning  autolysis  of  the  clot  by  the  intracellular  proteases  of 
the  muscle,  which  act  best  in  an  acid  medium,  but  proteoses  and 
peptones  cannot  be  found  in  such  muscle.  It  is  improbable  that  the 
degree  of  acidit}'  ever  becomes  so  high  that  the  myosin  is  redis- 
solved  through  a  conversion  into  acid  albumin  (syntonin),  as  was 
formerly  supposed,  v.  Fiirth  holds  that  the  re-solution  of  the  rigor  is 
caused  by  coagulation  of  the  proteins,  thus  reducing  this  hydrophilic 
tendency,  a  view  in  harmony  with  recent  developments  in  colloid 
chemistr}'." 

"Waxy"  degeneration  of  muscles,  although  usually  resulting  from 
the  action  of  toxic  substances,  is  entirely  different  from  cloudy  swell- 
ing, in  that  the  cytoplasm  has  become  homogeneous  and  not  granular. 
This  is  undoubtedly  due  to  the  increased  accumulation  of  acid  which 
takes  place  in  muscles  when  they  suffer  from  a  defective  oxygen  sup- 
ply, for  I  have  found  it  possible  to  produce  the  typical  appearance 
of  Zenker's  waxy  degeneration  by  letting  weak  solutions  of  lactic 
or  other  acids  act  on  muscle  fibers.  Even  excessive  stimulation  of 
muscles  was  found  to  be  sufficient  to  cause  waxy  degeneration,  the 
acid  being  formed  faster  than  it  can  be  removed. ^^ 

Muscles  showing  the  "reaction  of  degeneration"  have  been  anal3'zed  by 
Rumpf  and  Schumm,'^*  who  found  a  great  increase  in  the  fatty  matter,  which  was 
about  fifteen  times  the  normal  amount.  The  muscle,  deducting  the  fat,  showed 
a  loss  of  solid  matter  and  an  increase  of  water;  sodium  and  calcium  were  increased, 
potassium  decreased.  There  is  also  a  great  relative  increase  in  the  proportion  of 
phosphorus  bound  to  protein  in  muscles  which  have  atrophied  after  nerve  section, 
because  of  the  persistence  of  nuclear  and  loss  of  non-nuclear  elements  (Grund"®), 
but  there  is  little  change  in  the  proportion  of  mono-  and  di-amino  nitrogen.^' 
The  creatine  content  decreases  steadily  after  the  reaction  of  degeneration  is  first 
well  established.^* 

ATROPHY 

The  chemical  changes  of  simple  atrophy  have  not,  so  far  as  I  can 
find,  been  definitely  studied.  It  is  to  be  presumed,  in  view  of  the 
structural  changes,  that  analysis  of  atrophied  tissues  would  show  a 
relatively  high  nucleic  acid  and  collagen  content.  It  is  known  that  in 
atrophy  the  cell  lipoids  are  not  much  altered,  while  the  simpler  fats 
maj^  be  increased  in  parenchymatous  organs.  In  fatty  tissues,  of 
course,  the  fat  is  greatly  reduced,  its  place  being  partly  taken  by 
serum  (serous  atrophy  of  fat).     In  the  heart  muscle,  especially,  but 

^'  Corroborated  by  Lentz,  Zeit.  angew.  Chem.,  1912  (25),  1513;  and  Schwarz, 
Biochem.  Zeit.,  1912  (37),  35. 

"^  As  this  work  antedates  much  of  the  recent  work  on  the  influence  of  acids  of 
metabolic  origin  upon  the  swelling  of  cell  structures,  attention  may  be  called  to 
the  fact  that  a  preliminary  report  of  these  experiments  w-as  made  in  the  first 
edition  of  this  book,  written  in  1906. 

«5  Deut.  Zeit.  f.  Xervenheilk.,  1901  (20),  445. 

««  Arch.  exp.  Path.,  1912  (67),  393. 

"  Wakeman,  Jour.  Biol.  Chem.,  1908  (4),  137. 

"  Cathcart  et  al,  Jour.  Phvsiol.,  1918  (52),  70. 


396  RETROGRESSIVE  CHANGES 

also  to  a  less  extent  in  the  liver  and  kidney,  during  atrophy  there  is  an 
increased  pigmentation  (brown  atrophy)  apparently  consisting  of 
lipochromes  or  lipofuscins;  but  it  is  to  be  doubted  that  this  represents 
so  much  an  actual  increase  in  pigment  as  a  relative  increase  through 
loss  of  other  cellular  elements.  Atrophied  tissues  also  tend  to  undergo 
a  marked  compensatory  invasion  by  fatty  areolar  tissue  if  located  in 
contact  with  such  tissue;  e.  g.,  atrophy  of  muscles  after  nerve  section,®^ 
specific  muscular  dystrophies,  and  atrophy  of  the  pancreas.  In  the 
muscle  tissue  of  salmon  migrating  to  the  spawning  grounds  occurs  one 
of  the  most  marked  examples  of  atrophy,  and  Greene^^  has  found  that 
at  least  30  per  cent,  of  the  protein  lost  from  the  muscles  may  be  con- 
sidered as  stored  protein,  since  it  can  be  lost  without  injury  to  the 
muscle. 

Starvation,  of  course,  produces  typical  atrophic  changes  in  the 
tissues,  and  the  general  effects  on  metabolism  have  been  especially 
fully  worked  out  by  Benedict.'^"  The  structural  changes  in  parenchy- 
matous cells  are  described'^^  as  of  two  types;  first,  granular  changes 
and  vacuolization  of  the  cytoplasm,  resembling  the  effects  of  osmotic 
pressure  alterations;  second  and  later,  lysis  of  cytoplasm  with  also 
some  involvement  of  the  nuclei,  after  the  order  of  autolytic  changes. 
The  cell  walls  may  also  become  indistinct,  so  that  the  cells  resemble  a 
syncytium. '^^  In  the  atrophied  muscle  after  nerve  section  Wakeman®^ 
found  a  decrease  in  solids,  and  a  lowered  proportion  of  diamine  acids. 

Morse  has  studied,  by  experimental  methods,  the  question  of  the 
mechanism  involved  in  atrophy,  using  especially  the  involuting  tail 
of  the  tadpole  as  his  test  object.''^  He  beheves  that  autolysis  is  the 
primary  factor,  probably  induced  by  acidity  that  results  from  vascular 
occlusion.  The  involution  of  the  puerperal  uterus,  whether  it  can 
properly  be  called  atrophy  or  not,  seems  to  be  the  result  of  heightened 
autolysis,  the  products  of  which  are  excreted  quantitatively  in  the 
urine.''*  Bradley"  calls  attention  to  the  fact  that  atrophy  occurs 
commonly  under  conditions  of  reduced  blood  supply,  which  implies 
partial  asphyxia  and  a  resulting  tendency  to  local  excess  of  H-ions, 
which  would  favor  autolysis.  Conversely,  hypertrophy  is  observed 
with  abundant  blood  supply  which  tends  to  keep  the  reaction  of  the 
tissues  so  low  in  H-ions  that  autolysis  is  held  at  a  minimum. 

CLOUDY  SWELLING'6 

The  characteristic  appearance  of  organs  the  seat  of  cloudy  swell- 
ing, which  is  frequently  likened  to  a  "scalded"  appearance,   sug- 

69  Jour.  Biol.  Chem.,  1919  (39),  435. 

'">  Carnegie  Inst.  Publ.,  1915,  No.  203. 

"  Cesa-Bianchi,  Frankf.  Zeit.  Path.,  1909  (3),  723. 

"  Morgulis,  Howe  and  Hawk,  Biol.  Bull.,  1915  (28),  397. 

"  Biol.  Bull.,  1918  (34),  149. 

''*  Hlemons,  Bull.  Johns  Hopkins  Hosp.,  1914  (25),  195. 

"^  Jour.  Biol.  Choin     1910  (25),  201. 

"  Review  of  general  features  by  Landsteiner,  Ziegler's  Beitr.,  1903  (33),  237. 


CLOUDY  SWELLING  397 

gests  that  the  change  consists  in  a  coagulation  of  the  cell  proteins, 
which  idea  is  supported  by  the  similarity  of  the  niicTOscopic  changes 
observed  in  the  cells  and  the  earliest  microscopic  changes  observed  in 
cells  after  heating  gently  to  about  their  maximum  thermal  point. 
On  the  other  hand,  the  granules  in  cloudy  swelling  are  generally  de- 
scribed as  being  soluble  in  dilute  acetic  acid  and  dilute  KOH,  which 
indicates  that  they  are  not  the  result  of  ordinary  heat  coagulation. 
If  we  bear  in  mind,  however,  that  cloudy  swelling  probably  does  not 
represent  one  single  change,  it  may  be  possible  to  arrive  at  some 
understanding  of  the  chemical  changes  that  occur  in  the  process. 
Albrecht''^  considers,  with  good  reason,  that  we  may  have  a  granular 
appearance  of  cells  which  is  simply  an  exaggeration  of  the  normal 
granular  structure,  and,  although  it  may  be  observed  in  tissues  mod- 
erately affected  by  toxins,  or  in  starvation,  or  in  transitory  anemia, 
the  change  is  still  to  be  looked  upon  as  little  more  than  physiological 
in  response  to  stimuli  and  overwork.  Such  a  "cloudy  swelling"  may 
also  occur  in  cells  in  the  beginning  of  autolysis,  or  simply  under  the 
influence  of  salt  solution.  If  the  injury  is  greater,  however,  as  in 
profound  sepsis,  or  extreme  local  anemia,  the  granules  becomes  coarser, 
less  soluble  in  acetic  acid  and  KOH,  and  droplets  resembling  "myelin" 
make  their  appearance.  If  the  injury  is  still  more  severe,  true  coagu- 
lation of  the  granules  occurs,  and  they  become  insoluble,  the  fatty 
droplets  become  more  prominent,  and  the  cell  reaches  a  condition 
that  may  with  propriety  be  termed  necrosis  or  fatty  degeneration,  or 
both.  There  is  no  very  sharp  line  separating  necrosis  and  cloudy 
swelling,  especially  if  we  consider  only  the  changes  in  the  cytoplasm. 
In  the  earliest  stages  the  granules  are  perhaps  due,  in  some  cases,  to 
simple  aggregation  of  the  colloids,  without  the  development  of  a  true 
coagulation,  and  so  the  granules  are  still  soluble.  Possibly  bacterial, 
toxins  may  also  cause  soluble  precipitates,  but  this  does  not  appear 
to  have  been  established.  Halliburton  has  shown  that  temperatures 
that  may  be  reached  in  high  fevers  can  cause  turbidity  in  solutions 
of  cell  proteins,  and  hence  heat  precipitation  may  be  partly  responsi- 
ble for  the  turbidity  of  cells  in  cloudy  swelling,  but  it  is  doubtful  if 
the  granules  thus  formed  would  be  soluble  in  acetic  acid.  A  careful 
discussion  of  the  character  and  characteristics  of  this  process  is  given 
by  Bell,"^  wdio  concludes  that  the  term  cloudy  swelling  is  sound  only 
as  a  gross  description,  since  microscopically  the  cells  may  be  found  to 
show  albuminous  granules,  or  fatty  metamorphosis  or  simple  edema. 
When  present,  the  granules  are  of  unknown  nature — they  are  not 
identical  with  Altmann's  granules,  although  Aschoff  and  Ernst^^ 
both  consider  that  many  of  them  are  derived  from  the  mitochondria. 
An  enormous  number  of  granules  may  be  present  in  the  renal  cells 

"  Verb.  Deut.  Path.  Gesell.,  1903  (6),  63. 

'8  Jour.  Amer.  Med.  Assoc,  1913  (61),  455. 

"  Verb.  Deut.  Path.  Gesellsch.,  1914  (17),  43  and  103. 


398  RETROGRESSIVE  CHANGES 

without  demonstrable  impairment  of  function.^"  They  may  disap- 
pear dm-ing  acute  infections,  and  they  bear  no  constant  relation  to 
fatty  changes. 

We  may  speak  with  more  assurance  concerning  the  swelling  of  the 
cell,  and  attribute  it  to  an  edema  of  the  cell  contents,  it  having  been 
shown  that  in  cloudy  swelhng  the  water  content  of  the  organs  is  in- 
creased.^^ This  might  be  produced  by  a  rise  in  osmotic  pressure  due 
to  abnormally  rapid  splitting  of  proteins  with  incomplete  oxidation  of 
the  substances  formed,  which  results  in  formation  of  many  crj^stalloid 
molecules  with  high  total  osmotic  pressure,  from  a  smaller  number  of 
colloid  molecules  with  almost  no  osmotic  pressure.  It  has  frequently 
been  shown  that  the  cell-walls  do  not  lose  their  semipermeable 
character  until  the  death  of  the  cell  occurs;  hence  in  cloudy  swell- 
ing water  diffuses  in  much  more  rapidly  than  the  crystalloids  can 
diffuse  out,^^  causing  a  hydropic  swelling.  This  hypothesis  is  sup- 
ported by  the  observations  of  Cesaris  Demel,^^  who  found  that  by 
modifying  the  osmotic  conditions  of  the  cells,  particularly  epithelial 
cells,  he  could  closely  reproduce  many  of  the  characteristic  features 
of  parenchymatous  degeneration.  It  is  possible,  also,  that  too  high 
concentration  of  crystalloids  within  the  cells  may  be  a  factor  in  the 
precipitation  of  the  cell  colloids.  In  view  of  the  fact  that  in  the 
earliest  stages  of  autolysis,  histologic  and  microscopic  changes  closely 
resembling  those  of  cloudy  swelhng  are  pronounced,  and  that  organs 
the  seat  of  cloudy  swelling  notoriously  undergo  autolysis  with  extreme 
rapidity  after  death, ^"^  we  may  also  consider  that  this  process  is  possibly 
in  part  responsible  for  the  change  of  ordinary  intra  vitam  cloudy 
swelling.  The  appearance  of  fine  granules  of  lipoid  substance^""  (myelin 
or  "protagon")  in  cells  during  autolysis  and  during  cloudy  swelling 
is  in  support  of  this  idea,  and  chemical  analysis  of  organs  showing  cloudy 
swelling  gives  definite  evidence  of  autolytic  decomposition  of  the  pro- 
teins and  an  increase  in  the  water  content. ^^  Presumably  this  increase 
in  water  is  the  cause  of  the  lowered  specific  gravitj'-  of  organs  exhibiting 
parenchymatous  degeneration.^^  Landsteiner,  through  his  studies  of 
cloudy  swelling  in  human  material,  also  came  to  the  conclusion  that 
autolysis  is  an  important  element  in  its  production. 

Martin  H.  Fischer^*^  applies  the  principles  of  colloidal  chemistry 
to  the  problem  and  concludes  that  the  changes  of  cloudy  swelling 

80  Shannon,  .lour.  Lab.  Clin.  Med.,  191G  (1),  541. 

81  Schwenkenbecher  and  Ingaki,  Arch.  exp.  Path.  u.  Phann.,  1906  (55),   203. 

82  See  introductory  chapter  concerning  osmosis;  also  discussion  of  edema. 

83  Lo  Sperimentale,  1905;  Cent.  f.  Path.,  1905  (IG),  613. 

84  See  Medigreceanu,  Jour.  Exp.  Med.,  1914  (19),  309. 

s^Orglcr,  Virchow's  Arcli.,  1904  (176),  413;  Hess  and  Saxl,  ibid.,  1910  (202), 
149. 

86  Verh.  Deut.  Path.  Gesell,  1903  (6),  76. 

87  See  Olsho,  Arch.  Int.  Med.,  1908  (2),  171. 

88  "Oedema and  Nephritis,"  New  York,  1915,  p.  455; also  Zeit.  Chcm.  u.  Indust. 
Colloide,  1911  (8),  159. 


CLOUDY  SWELLING  399 

may  be  ascribed  to  acids  developed  in  the  cell.  It  is  of  significance 
that  Chambers^'-*  has  found  that  even  slight  mechanical  injury  of  iso- 
lated cells  under  the  microscope  produces  a  demonstrable  acidity  in 
the  protoplasm.  Electro-negative  proteins  in  the  cell  are  precipitated 
by  weak  concentrations  of  acids,  forming  the  granules  in  the  cells, 
which  can  be  dissolved  again  by  a  stronger  concentration  of  acid  as  in 
the  characteristic  clearing  of  granular  cells  by  acetic  acid.  The 
swelling  is  explainable  by  the  increased  affinity  for  water  of  other  cell 
proteins  under  the  influence  of  acids.  This  theory  is  supported  by  good 
experimental  evidence  and  has  much  in  its  favor,  the  chief  question 
being  whether  the  blood  cannot,  under  ordinary  conditions  of  circula- 
tion, furnish  sufficient  neutralizing  salts  to  prevent  acidification  in 
the  cells  to  cause  cloudy  swelling. 

s^Amer.  Jour.  Physiol.,  1917  (43),  1. 


CHAPTER  XVI 

RETROGRESSIVE  CHANGES  (Continued) 

Fatty,   Amyloid,   Hyaline,    Colloid,    and    Glycogenic    Infiltration   and 

Degeneration 

FATTY  METAMORPHOSIS 

In  1847,  in  the  first  number  of  his  Archiv,  Virchow  divided  the 
forms  of  fatty  changes  that  may  occur  in  pathological  conditions 
into  two  groups — "infiltration"  and  "degeneration" — -a  division 
that  has  since  become  classical.  By  infiltration  he  indicated  the  ex- 
cessive accumulation  of  fat  in  the  cells  in  the  form  of  large  droplets, 
without  destruction  of  the  nucleus  or  irreparable  damage  to  the  cells, 
and  by  the  use  of  the  term  infiltration  he  implied  his  belief  that  the 
fat  entered  the  cell  from  without.  When  the  fat  remained  in  the  form 
of  fine  droplets  and  the  cell  became  much  disintegrated,  Virchow 
considered  that  the  fat  was  derived  from  the  breaking  down  of  the  cell 
proteins,  and  hence  the  process  was  considered  to  be  a  fatty  degenera- 
tion of  the  protoplasm.  Since  that  time  scarcely  any  other  subject 
in  pathology  has  been  more  warmly  discussed  than  that  of  the  origin 
of  the  fat  in  fatty  degeneration,  and  an  appalling  amount  of  literature 
has  accumulated  concerning  the  questions  involved.  It  will  be  im- 
possible to  give  more  than  the  essential  facts  that  have  been  developed, 
referring  the  reader  for  the  full  details  of  the  discussion  and  evidence 
to  the  numerous  compilations  of  literature,  particularly  those  of  Rosen- 
feld,'  and  to  the  original  articles  cited  in  the  text. 

I  "Fat  Formation,"  Ergebnisse  der  Physiol.,  Abt.  1,  1902  (1),  651;  ibid.,  1903 
(2),  50.  Also  see  discussion  in  the  Verh.  Deut.  Path.  Gesell.,  1904  (6),  37-108, 
and  the  review  by  Leathes  in  his  "Problems  in  Animal  Metabolism,"  1906,  pp 
71-121,  and  "The  Fats  "  Monographs  on  Biochemistry,  London,  1910;  von  Fiirth, 
"Chemistry  of  Metabolism,"  Amer.  Transl.,  New  York.  1916.  Concerning  theor- 
ies of  role  of  lipase  in  fat  metabolism  see  Chap.  iii.  Other  reviews  of  literature  on 
pathological  fat  formation  by  Christian,  Johns  Hopkins  Hosp.  Bull,  1905  (16), 
1;  Lohlein,  Virchow's  Arch.,  1905  (180).  1;  Pratt,  Johns  Hopkins  Hosp.  Bull. 
1904  (15;,  301  (particular  reference  to  heart);  Wohlgemuth,  Handbuch  d.  Bio- 
chem.,  1909,  III  (1),  150;  Magnus-Levy  and  Meyer,  ibid.,  1910,  IV  (1),  445; 
Dietrich,  Ergebnisse  der  Pathol.,  1909,  XIII  (2),  283.  Concerning  Obesity  see 
V.  Bergmann,  Handbuch  d.  Biochem.,  1910,  IV  (2),  208.  Later  references  of  im- 
portance cited  in  the  text. 

400 


FATTY  METAMORPHOSIS  401 

Physiological  Formation  of  Fat 

Concerning  the  normal  formation  of  fat  we  may  summarize  the  evidence  as 
follows : 

(1)  A  large  proportion  of  the  fat  of  the  body  comes  from  the  fat  taken  in  the 
food,  as  also  does  the  fat  of  the  milk.  This  can  be  shown,  as  Rosenfeld  particu- 
larly demonstrated,  by  starving  an  animal  until  it  is  as  free  from  fat  as  possible, 
then  feeding  with  a  large  amount  of  some  fat  that  is  of  a  type  different  from  that 
normally  found  in  the  animal;  the  new  fat  that  it  then  laid  up  in  the  fat  depots  of 
the  animal  will  partake  of  the  characters  of  the  fat  given  in  the  food.  In  case  the 
animal  is  lactating,  the  milk-fat  will  also  resemble  the  fat  of  the  food.  As  a 
matter  of  fact,  the  body  fat  is  not  of  constant  composition,  even  in  the  same 
individual;  it  varies  greatly  with  age,  having  much  less  olein  in  infancy  than  in 
later  years,  varying  somewhat  in  composition  in  the  different  fat  depots  in  the 
same  body,  and  apparently  being  more  or  less  modified  by  diet. 

(2)  Fat  may  also  be  formed  from  carbohydrates.  According  to  Rosenfeld,  this 
fat  differs  from  the  fat  formed  on  mixed  diet  in  having  less  olein  in  proportion  to 
the  palmitin  and  stearin,  and  it  is  deposited  particularly  in  the  subcutaneous  and 
mesenteric  tissues  rather  than  in  the  liver.  Man  does  not  seem  to  form  fat  readily 
from  carbohydrates,  but  rather  burns  them  to  protect  his  proteins;  on  the  other 
hand,  swine  and  geese  readily  form  fat  from  carbohydrates.  As  the  fatty  acid 
radicals  of  ordinary  fat  (C18H36O2,  Ci6H.-;202,  C1SH34O2),  are  much  larger  than  the 
carbotiydrate  radicals,  a  process  of  synthesis  must  be  involved  in  the  formation  of 
fat  from  carbohydrates.^ 

(3;  Proteins  are  a  possible  source  of  fat,  but  it  has  not  been  established  that 
they  are  either  a  common  or  an  important  source  of  fat  in  either  physiological  or 
pathological  conditions,  or,  indeed,  that  they  really  ever  do  form  fat.  Upon  this 
statement  rests  our  present  tendency  to  refute  the  long-cherished  conception  of 
fatty  degeneration  as  a  true  degeneration  of  cell  proteins  into  fat,  as  suggested  by 
Virchow.  This  view  was  supported  by  the  earlier  work  of  ^"oit  and  his  school,  who 
believed  that  they  had  demonstrated  that  animals  could  form  fat  from  protein 
food,  and  their  work  was  for  a  long  time  accepted  as  correct.  Later  Pfliiger  and 
his  pupils  pointed  out  what  seem  to  have  been  essential  errors  in  these  investigations, 
and,  after  much  discussion  and  experimentation,  the  majority  of  physiologists  now 
support  the  view  advanced  in  the  sentence  opening  this  paragraph.  Since  proteins 
contain  carbohydrate  groups,  and  since  fats  can  be  formed  from  carbohydrates, 
the  possibility  of  the  formation  of  fats  from  the  proteins  in  this  indirect  way 
cannot  be  denied.  It  is  also  possible  that  the  nitrogen-containing  groups  may  be 
split  out  of  the  amino-acids  of  the  protein  molecule,  and  that  the  non-nitrogenous 
residues  can  then  be  built  up  into  fatty  acid  molecules  as  large  as  the  molecules  of 
stearic,  palmitic,  and  oleic  acids;  but  we  have  no  proof  that  either  of  these  processes 
occurs  in  the  normal  cell  or  in  the  cell  that  is  undergoing  degeneration.  Atkinson 
and  Lusk'  have  obtained  evidence  of  some  fat  formation  from  meat  fed  to  a  dog, 
but  this  was  only  slight  and  obtained  with  difficulty. 

Pathological  Fat  Accumulation 

For  a  long  time  fatty  degeneration  was  looked  upon  as  one  of  the 
chief  evidences  that  fat  was  formed  directly  from  protein,  for  the 
cell  protoplasm  seemed,  morphologically,  to  be  changed  directly  into 
fat  in  this  process.  Additional  support  was  also  claimed  from  the 
supposed  increase  in  fat  in  the  ripening  of  cheese;^  from  the  forma- 
tion of  abundant  fat  by  maggots  living  in  fat-poor  blood  or  fibrin; 
and  by  the  apparent  conversion  of  proteins  into  fatty  acids  and  soaps 

2  This,  Magnus-Levy  suggests,  may  be  accomplished  through  lactic  acid  which 
is  formed  from  sugar,  and  then,  after  reduction  to  an  aldehyde,  several  of  these 
molecules  are  combined  into  the  higher  fatty  acid.     See  Leathes,  loc.  cit.,  p.  82. 

3  Proc.  Natl.  Acad.  Sci.,  1919  (5),  2-16. 

*  Even  the  increase  of  fat  in  ripening  cheese  is  doubtful  (Nierenstein,  Proc. 
Royal  Soc,  B.,  1911  (83),  301;  Kondo,  Biochem.  Zeit.,  1914  (59),  113). 

26 


402  RETROGRESSIVE  CHANGES 

in  the  postmortem  change,  adipocere.  But  it  has  now  been  well  es- 
tabhshed  that  there  is  no  true  conversion  of  protein  into  fat  in  the  fatty- 
degeneration  produced  experimentally  by  poisoning  with  phosphorus, 
etc.,^  and  the  other  supposed  instances  of  fat-formation  above  cited' 
have  been  discredited  by  various  methods  which  it  will  not  serve  our 
purpose  to  discuss  here,  beyond  mentioning  that  one  of  the  chief 
sources  of  error  Hes  in  the  fact  that  many  fungi  and  bacteria^  can 
form  fat  from  protein. 

It  having  been  rendered  probable  that  fat  was  not  formed  by  dis- 
integration of  the  protein  of  the  degenerating  cells,  it  remained  to 
determine  what  the  source  of  the  fat  observed  in  the  cells  under  patho- 
logical conditions  might  be,  and  this  part  of  the  problem  has  been 
largely  cleared  up  by  Rosenfeld.  This  investigator  proceeded  as  fol- 
lows: Animals  were  starved  until  they  were  extremely  poor  in  fat, 
then  fed  upon  easily  identified  foreign  fats,  such  as  mutton  tallow 
(which  has  a  liigh  melting-point  and  can  combine  with  httle  iodin) 
or  linseed  oil  (which  has  a  low  melting-point  and  can  combine  with 
much  iodin).  The  animals  under  these  conditions  laid  up  in  their 
fat  depots,  including  the  liver  as  well  as  the  subcutaneous  tissues, 
large  quantities  of  these  foreign  fats.  By  starving  again  for  a  few 
days  the  foreign  fat  was  removed  from  the  liver,  leaving  still  a  large 
amount  in  the  other  storehouses,  and  the  animals  were  then  poisoned 
with  phosphorus  or  other  poisons  that  cause  a  typical  fatty  degener- 
ation of  the  liver  and  other  viscera.  When  the  fat  was  extracted  from 
the  fatty  hver  of  these  animals,  it  was  found  that  the  new  fat  that 
had  appeared  in  the  liver  during  the  process  was  not  normal  dog  fat 
(which  it  should  have  been  if  formed  by  degeneration  of  the  cell  pro- 
teins), but  was,  in  part,  of  the  same  type  as  the  foreign  fat  which 
the  animals  had  deposited  in  their  subcutaneous  tissues  and  other  fat 
storehouses.  Furthermore,  it  was  found  that  animals  starved  to  an 
extremely  low  fat  content  do  not  develop  the  typical  fatty  liver  of 
phosphorus-poisoning,  a  fact  which  Lebedeff  had  already  noted  in  a 
ease  of  phosphorus-poisoning  in  an  emaciated  patient.  Of  similar 
significance  is  the  fact  that  in  fatty  human  livers  the  iodin  number, 
normally  high,  falls  as  the  amount  of  fat  increases  until  it  is  approxi- 
mately that  of  adipose  connective  tissue.'^  Therefore,  it  seems  evident 
that  the  fat  accumulating  iii  the  liver  during  fatty  degeneration  is  not 
derived,  as  Virchow  thought,  through  a  tJ-ansformation  of  cell  proteins  into 
fat,  but  rather  is  an  infiltrated  fat  brought  in  the  blood  fro^n  the  fat  deposits 
of  the  body  to  the  disintegrating  organ.  This  work  has  since  been  corro- 
borated and  extended  by  many  observers,  and  its  correctness  can  now 

^See  Taylor,  Jour.  Exp.  Med.,  1899  (4),  399;  Shibata,  Biochem.  Zeit.,  1911 
(37),  345. 

"  See  Beebe  and  Buxton,  Amer.  Jour,  of  Physiol.,  1905  (12j,  466;  Slosse,  Arch. 
Internat.  Physiol.,  1904  (Ij,  348. 

^  Leathes,  Lancet,  Feb.  27,  1909;  Hartley  and  Mavrogordato,  Jour.  Path,  and 
Bact.,  1908  (12;,  371;  Jackson  and  Pearce,  Jour.  Exp.  Med.,  1907  (9),  578. 


FATTY  METAMORPHOSIS  403 

hartlly  be  questioned.'*  "Fatty  degeneration/'  tliercforc,  at  least  in 
some  cases,  differs  from  "fatty  infiltration"  chiefly  in  the  fact  that 
in  the  former  the  process  is  associated  with  serious  injury  to  the  cell, 
caused  by  the  action  of  toxins  or  loss  of  nutrition,  while  in  the  latter 
the  cell  is  not  seriously  injured  and  is  capable  of  returning  to  its  nor- 
mal condition  whenever  the  fat  is  removed. '■* 

Fatty  "Degeneration"  without  Infiltration. — By  showing 
that  new  fat  in  fatty  livers  is  infiltrated  fat,  Rosonfeld  did  not  entirely 
clear  up  the  subject,  for,  in  the  course  of  his  analyses  of  organs  that 
were  micro-  or  macro-scopically  the  seat  of  fatty  degeneration,  he 
found  that  there  is  not  always  any  correspondence  between  the  amount 
of  fat  that  seems  to  be  present,  as  determined  by  microscopic  methods, 
and  the  amount  that  chemical  analysis  shows  to  be  present.  This 
is  particularly  true  of  the  kidney.  Thus,  the  amount  of  fat  and 
lipoids,  or  lipins,  present  in  normal  kidneys  (dog)  was  found  to  vary 
between  18.5  per  cent,  and  29.12  per  cent,  of  the  dry  weight,  the  aver- 
age being  21.8  per  cent.;  whereas,  after  producing  a  typical  "fatty 
degeneration"  by  means  of  phosphorus  and  other  poisons,  the  lipin 
content  was  still  found  to  be  between  16.9  per  cent,  and  22.6  per  cent.^" 
In  all  instances  the  amount  of  lipins  in  kidneys  showing  typical  fatty 
degeneration  under  the  microscope  was  found  equal  to  or  less  than 
the  normal  amount — it  was  never  increased.  The  same  conditions 
were  found  to  obtain  in  human  kidneys  that  showed  fatty  metamor- 
phosis. Microscopic  examination  of  specimens  stained  with  the  speci- 
fic fat  stains,''  therefore,  gives  no  indication  of  the  amount  of  fat 

8  Schwalbe  (Verh.  der  Deut.  Path.  Gesell.,  1903  (6),  71)  claims  that  in  a  sim- 
ilar way  iodin  compounds  of  fat  can  be  demonstrated  to  be  transported  into  the 
fatty  organs.  His  analyses  were  merely  qualitative  and  by  quantitative  deter- 
minations 1  was  unable  to  corroborate  his  conclusions  (Zeit.  f.  physiol.  Chem., 
1905  (45),  412). 

^  A  striking  proof  of  the  lack  of  injury  associated  with  fatty  infiltration  is 
shown  by  the  fatty  infiltration  frequently  seen  in  the  liver,  especially  of  alcoholics, 
in  which  it  may  be  difficult  to  find,  microscopicallj',  anj'  cell  cytoplasm  because 
of  the  fat,  the  tissue  looking  like  fatty  areolar  tissue;  and  j^et  there  may  be  no 
clinical  evidence  whatever  that  the  liver  fimotion  has  been  impaired  by  the  process. 

1"  Concerning  the  normal  intracellular  fats  see  introductory- chapter. 

"  Fat-staining  involves  several  principles  of  interest  in  this  connection.  (See 
reviews  by  BuUard,  Jour.  Med.  Res.,  1912  (27),  55  and  Escher,  Corrlil.  Schweizer 
Aertze,  1919  (49),  1609.)  Osmic  acid  (OsO^),  the  longest  used  for  this  purpose,  is 
reduced  to  OsOi  by  oleic  acid,  imparting  a  black  or  dark-brown  color  to  the  fat; 
but  it  does  not  stain  staurated  fatty  acids,  such  as  palmitic  or  stearic  acid.  Thus, 
Christian  found  in  pneumonic  exudates  fat  that  stained  by  other  methods  but  not 
by  osmic  acid,  apparently  because  it  contained  no  oleic  acid  (Jour.  Med.  Research, 
1903  (10),  109).  Sudan  111  and  scarlet  II  {fat  inmccau)  are  two  sj-nthetic  dyes 
which  stain  fat  in  a  purely  physical  way,  entering  and  remaining  in  the  fat-droplets 
because  they  are  much  more  soluble  in  fat  than  they  are  in  water  or  alcohol. 
(Fulh^  discussed  by  Michaelis  (who  introduced  scarlet  R)  in  Virchow's  Arch.,  1901 
(164),  263;  and  by  Mann,  "Physiological  Histology,"  p.  306.)  These  stains  have 
the  advantage  of  staining  all  sorts  of  fats  and  not  staining  other  substances  that 
may  reduce  osmic  acid.  Fatty  acids  and  soaps  may  be  stained  with  copper  acetate, 
which  forms  a  green  copper  salt,  and  thus  be  distinguished  from  fats  (Benda,  Vir- 
chow's Arch.,  1900  (161),  194).  J.  Lorrain  Smith  (Jour.  Path,  and  Bact.,  1907 
(12),  1)  has  introduced  as  a  fat  dye,  Nile  blue  sulphate,  which  forms  a  blue  salt 
with  free  fatty  acids,  while  neutral  fats  are  stained  red  bj'  the  oxazone  base. 


404  RETROGRESSIVE  CHANGES 

contained  in  a  degenerated  kidney.  A  pathologic  kidney  containing 
16  per  cent,  of  lipins  ( 18  per  cent,  is  about  the  average  amount  in  normal 
human  kidneys)  may  show  extreme  "fatty  degeneration"  under  the 
microscope,  whereas  another  kidney  may  contain  as  much  as  23  per 
cent,  of  hpins,  yet  not  show  any  fat  whatever  by  staining  methods. 
The  explanation  of  this  remarkable  discrepancy  is  as  follows: 
Every  tissue  and  organ  seems  to  contain  a  greater  or  less  amount  of 
lipins,  varying  from  5  per  cent,  to  20  per  cent,  of  the  total  dry  weight 
of  the  organ  in  the  case  of  most  of  the  important  tissues,  yet  this  is 
usually  held  in  such  a  form  that  it  cannot  be  stained  by  any  stains 
available  for  the  purpose.  Thus  in  the  kidneys,  as  before  remarked, 
we  may  have  as  much  as  23  per  cent,  of  lipins  present  and  yet  be  unable 
to  stain  any  of  it  by  ordinary  methods.  The  greater  part  of  this  seems 
to  be  essential  to  the  cell,  for  it  cannot  be  removed  by  the  most  extreme 
starvation;  e.  g.,  the  liver  of  the  most  emaciated  dogs  may  contain 
10  per  cent,  to  20  per  cent,  of  fatty  substances.  Furthermore,  the 
same  resistance  is  shown  by  part  of  the  fat  to  extraction  with  ether. 
A  certain  proportion  of  the  fat  can  be  extracted  readily  in  twenty- 
four  hours  or  less  by  ether,  but  after  this  time  no  more  can  be  made 
to  leave  the  tissues.  Apparently  the  rest  of  the  fat  is  held  in  a  com- 
bination that  is  insoluble  in  ether,  and  a  large  proportion  of  this  fixed 
material  is  not  simple  fat,  but  lecithin,  cholesterol,  and  compounds  of 
these  lipoids.  It  has  also  been  demonstrated  that  fatty  acids  can 
combine  with  amino-acids  to  form  compounds  (lipo-peptids)  very 
similar  in  their  properties  to  these  "masked"  fats.^^  By  digesting 
the  tissue  for  a  short  time  by  pepsin,  however,  the  fixed  lipins  become 
freed,  so  that  they  can  then  be  readily  dissolved  out  in  ether.  We  see, 
therefore,  that  much  of  the  fat  of  normal  cells  is  so  firml}-  combined 
that  it  cannot  be  dissolved  in  ether,  and  under  normal  conditions  all, 
or  nearly  all,  of  it  cannot  be  stained.  (This  applies  particularly  to 
the  parenchymatous  organs;  the  fat  of  the  areolar  tissue  is  all  readily 
extracted — Taylor.)  By  the  use  of  Ciaccio's  method  for  microscopic 
demonstration  of  intracellular  lipoids,  BelP''  has  been  able  to  demon- 
strate in  those  cells  that  are  fat-free  by  ordinary  methods  sufficient 
lipoidal  material  to  account  for  the  normal  "invisible  fat,"  which  is 
probably  identical  with  the  "liposomes."  But  when  pathological 
changes  in  the  cells  result  in  decomposition  of  the  cell  protein  through 
autolysis,  or  produce  physical  changes  in  the  colloids  that  hold  the 
lipins  emulsionized,  part  of  this  normally  invisible  fat  is  set  free,  and, 
becoming  visible,  "phanerosis,"  ^'^  produces  the  so-called  "fatty  degen- 
eration." This  explains  the  observations  of  Rosenfeld,  cited  above, 
that  kidneys  may  show  much  fat  to  the  naked  eye  and  microscopically, 
when  they  actually  contain  even  less  than  normal  amounts  of  fat.     Tay- 

12  Bondi,  Biochem.  Zcit.,  1909  (17),  543. 

"Internat.  Monats.  Anat.  u.  Physiol.,  1911  (28),  297;  Jour.  Med.  Res.,  1911 
(24),  539. 

'*  Klein perer,  Deut.  med.  Woch.,  1909  (35),  89. 


FATTY  METAMORPHOSIS  405 

lor^^  advanced  this  explanation,  and  supported  it  experimentally  by 
showing  that  during  fatty  degeneration  this  protected  fat  actually  is 
liberated,  some  two-thirds  becoming  ether-soluble  in  an  experiment 
performed  with  phosphorus-poisoned  frogs.  Mansfeld""'  also  found 
that  in  animals  poisoned  with  phosphorus,  the  proportion  of  fat  which 
is  present  in  a  form  free  from  protein  union  in  both  blood  and  viscera, 
is  increased,  while  the  firmly  bound  fat  is  decreased.  As  further 
support  may  be  mentioned  the  fact  that  organs  undergoing  experi- 
mental autolysis  show  microscopically  an  apparently  typical  fatty 
degeneration,  although  analyses  show  that  no  actual  increase  in  fat 
occurs.  ^^ 

Relation  of  Anatomical  to  Chemical  Changes. — From  the 
facts  brought  out  in  these  various  experiments  we  must  consider 
that  the  anatomically  estabhshed  condition  of  "fatty  degeneration" 
represents  either  or  both  of  two  conditions:  (1)  It  may  result  from 
an  increase  in  the  normal  quantity  of  fat  in  an  organ  undergoing  paren- 
chymatous degeneration,  through  an  infiltration  of  fat  from  the  out- 
side; this  is  particularly  true  of  the  fatty  degeneration  of  the  hver, 
presumably  because  the  hver  normally  receives  the  relatively  saturated 
body  fats  to  work  them  over  into  the  more  labile  desaturated  fats;  (2) 
there  may  be  no  increase  in  the  total  amount  of  fat,  but  the  invisible 
fat  becomes  visible  through  autolj^sis  or  hydration  changes  in  the  cell 
proteins.  Thus,  Bainbridge  and  Leathes'^  found  that  after  ligation 
of  the  hepatic  artery  there  is  a  marked  fatty  degeneration  of  the  hver, 
without  an  increase  in  the  amount  of  fat  according  to  analysis.  (3) 
Finally,  of  course,  both  factors  may  occur  together.  Of  these  various 
forms,  in  only  the  first  would  the  chemist  consider  the  organ  "fatty, " 
although  from  a  morphological  standpoint  the  second  form  is  entitled 
to  rank  as  a  true  "fatty  degeneration,"  and  the  form  that  will  occur 
seems  not  to  depend  upon  the  cause  of  the  cell  injurj^,  but  rather  upon 
the  organ  under  consideration.  In  a  studj'-  of  the  relation  of  the  mor- 
phological to  the  chemical  changes  Rosenfeld^^  arrived  at  the  following 
results : 

Normal  human  hearts  contain,  on  an  average,  15.4  per  cent,  of  lip- 
ins;  the  hearts  showing  fatty  degeneration  contain  20.7  per  cent,  on  an 
average. -°     The    pancreas,    which  normally    contains   15.8-17.4  per 

'*  Jour.  Med.  Research,  1903  (9),  59. 

"  Pfluger's  Arch.,  1909  (129),  63. 

''  Dietrich,  Arb.  path.  Inst.  Tubingen,  1906  (5),  H.  3;  Hess  and  Saxl,  Virchow's 
Arch.,  1910  (202),  149;  Ohta,  Biochem.  Zeit.,  1910  (29),  1;  Shibata,  ibid.,  1911 
(31),  321.  The  significance  of  the  increase  of  lipins  observed  in  perfused  kidneys 
by  Gross  and  Vorpahl  is  made  doubtful  by  the  article  of  Underhill  and  Hendrix. 
Jour.  Biol.  Chem.,  1915  (22),  471. 

"  Bioehem.  Jour.,  1906  (2),  25. 

"  Berl.  klin.  Woch.,  1904  (41),587. 

^°  The  amount  of  phospho-lipins  in  the  heart  is  usually  nearly  constant,  but 
alimentary  fat  may  accumulate  in  the  myocardium  under  certain  conditions. 
See  Wegelin,  Berl.  klin.  Woch.,  1913  (50),  2125;  Bullard,  Amer.  Jour.  Anat., 
1916  (19),  1. 


406  RETROGRESSIVE  CHANGES 

cent.,  also  contains  an  increased  amount  when  showing  fatt}'  degenera- 
tion. The  liver,  however,  takes  on  by  far  the  greatest  amount  of  fat 
after  "steatogenetic"  poisons, ^^  and  the  microscopic  picture  usually 
gives  a  very  good  approximation  of  the  amount  of  lipins  it  contains. ^- 
Apparently  in  these  organs  any  excessive  fat  above  the  normal  is 
observable  microscopically,  although  the  normal  lipin  content  is  not, 
and  only  in  these  three  organs  could  Rosenfeld  find  an  actual  increase 
in  fat  after  poisoning  with  phosphorus,  etc.  It  would  seem,  on  the 
other  hand,  that  there  is  not  often  a  real  increase  in  the  fat  content  of 
the  "fatty"  kidney.^^  Normal  spleen  contains  14.2  per  cent,  of  lipins, 
and  lung  17.3  per  cent.,  but  in  both,  "fatty  degeneration"  results  in  a 
lowering  of  this  quantity.  Degenerations  in  the  nervous  tissue,  which 
Virchow  considered  the  best  evidence  of  the  conversion  of  protoplasm 
into  fat,  also  show  a  marked  decrease  in  lipins,  and  voluntary  muscle 
shows  no  increase  in  the  normal  quantity  after  poisoning.  In  general, 
these  experiments  support  the  contention  of  Taylor  concerning  the 
disclosure  of  the  invisible  fat  through  autolysis. -■*  An  explanation  of 
many  of  the  discrepancies  lies  in  the  newer  studies  on 

The  Relation  of  the  Lipoids  to  Fatty  Metamorphosis.-^ — Until 
within  a  few  years  the  significance  of  the  intracellular  lipoids  in  fatty 
degeneration  and  related  processes  was  not  appreciated,  beyond  the 

^1  In  fatty  livers  in  phosphorus-poisoning  the  amount  of  fat  may  reach  75  per 
cent,  of  the  dry  weight.  Accompanying  the  fat  increase  are  increase  in  water 
and  a  relative  or  absolute  decrease  in  proteins,  probably  due  to  cell  autolysis. 
In  acute  yellow  atrophy  a  similar  decrease  in  protein  occurs,  but  without  an  in- 
crease in  fat.     (See  v.  Starck,  Deut.  Arch.  klin.  Med.,  1884  (35),  481.) 

22  See  Helly  (Beitr.  path.  Anat.,  1914  (60),  1)  who  examined  100  human 
livers  which  showed  all  variations  in  microscopic  fat  content,  and  chemically 
from  7.36  to  74.43  per  cent,  of  lipins  (dry  weight).  He  found  that  there  was 
usually  a  good  correspondence  between  microscopic  appearance  and  analytic  re- 
sults, altho  some  marked  and  unexplained  discrepancies  were  observed.  Gener- 
ally the  fat  content  was  from  10  to  30  per  cent,  of  the  dry  weight,  with  19  to 
21  per  cent,  the  most  common  figures.  When  there  is  much  fat  present  in  the 
liver  the  fat  content  of  the  bile  is  increased  (Le  Count  and  Long,  Jour.  Exp.  Med., 
1914  (19),  234). 

^^  This  is  contradicted  by  Landsteiner  and  Mucha  (Cent.  f.  Path.,  1904  (15), 
752),  and  by  Lohlein  (Virchow's  Arch.,  1905  (180),  1)  and  Rosenthal  (Deut. 
Arch.  klin.  Med.,  1903  (78),  94),  but  is  supported  by  Orgler  {ibid.,  1904  (176), 
413),  and  Dietrich,  Verb.  Deut.  Path.  Gesell.,  1907  (11),  10.  See  also  the  later 
studies  by  Rosenfeld  on  the  effects  of  various  steatogenetic  poisons  on  different 
organs,  in  Arch.  f.  Exp.  Path.  u.  Pharm.,  1906  (55),  179  and  344.  It  is  probable 
that  the  truth  lies  between  the  opposing  views,  namely,  the  kidney  may  under 
some  conditions  take  up  fat  from  tlie  blood,  but  it  does  so  to  a  much  less  extent 
than  the  liver,  and  it  may  sometimes  show  marked  fatty  change  anatomically 
without  corresponding  increase  chemically. 

2''  Pieces  of  tissue  implanted  into  animals  may  show  a  perii)lieral  fatty  meta- 
morphosis or  infiltration,  yet  show  upon  analysis  a  decreased  fat  content  (Dietrich, 
Verb.  Deut.  Path.  Gesellsch.,  1905  (9),  212).' 

^f*  Literature  by  Leathes,  "The  Fats,"  London,  1910;  Bang,  Ergebnisse  der 
Physiol.,  1909  (8),  463,  also,  "Chemie  u.  Biochem.  d.  Lijjoide,"  Bergniann,  Wies- 
baden, 1911;  Kawanuu'a,  Virchow's  Arch.,  1912  (207),  4()9,  also  "Die  Cholester- 
inesterverfcttung,"  Fischer,  Jena,  1911;  Asclioff,  Zieglor's  Beitr.,  1909(47),!, 
also  Festschr.  f.  IJnna,  1911  p.  23;  SchuUz,  l<;rgel)nisse  d.  Pathol.,  1909  (XIII;), 
253;  Ilanes,  Bull.  Johns  Hopkins  Hosp.,  1912  (23),  77;  Anitschkow  and  Chala- 
tow.  Cent.  f.  Pathol.,  1913  (24),  1. 


FATTY  METAMORPHOSIS  407 

fact  that  in  most  organs  showing  fatty  changes  the  quantity  of  choles- 
terol and  lecithin  is  not  greatly  changed.  In  1902  Kaiserling  and 
Orgler  described  under  the  non-committal  name  of  "myelin"  certain 
intracellular  droplets  that  may  be  found  in  the  cells  of  the  normal 
adrenal  cortex,  and  in  amyloid  kidney's,  pneumonic  exudates,  tumor 
cells,  retrogressive  thymus  tissue,  corpus  luteum,  and  bronchial  secre- 
tions, and  which  differ  from  fat  in  being  doubly  refractile  (anisotropic) 
when  viewed  through  Nicoll  prisms,  and  in  staining  but  shghtly  gray 
with  osmic  acid,  although  taking  up  other  fat  stains  well. 

As  explained  in  Chapter  i,  the  myelins  are  probably  mixtures  of 
Upins,  cholesterol-esters  being  prominent,  and  in  many  conditions  in 
which  fat-like  vacuoles  are  prominent  in  cells,  leading  to  the  diagnosis 
of  fatty  degeneration,  these  substances  are  responsible,  presumably 
having  been  liberated  from  combination  with  the  cell  proteins  in  some 
cases,  in  others  actually  being  increased  in  the  cell.  This  condition, 
which  Aschoff  refers  to  as  a  cholesterol-ester  fatty  metamorphosis,  is 
especially  seen  in  the  parenchyma  cells  derived  from  the  urogenital 
anlage — ^that  is,  the  adrenal  cortex,  kidnej",  testicle  and  corpus  lu- 
teum. Aschoff  states  that  doubly  refractile  droplets  can  be  formed 
by  lecithin  and  phosphatids  generally,  oleates,  cholesterol  esters,  cho- 
lesterol v^hen  dissolved  in  phosphatids  or  fats  or  fatty  acids,  as  well 
as  by  cholesterol  esters  dissolved  in  fats.  Of  these  the  most  im- 
portant quantitatively  is  the  cholesterol  ester  group, -*^  and  the  anal- 
yses of  Windaus  have  shown  that  in  pathological  processes  the  increase 
is  much  greater  in  the  cholesterol  esters  than  in  the  free  cholesterol. 
Cholesterol  compounds  stain  different!}^  from  neutral  fats,  being  more 
yellow  than  red  with  sudan  III,  and  grajash  rather  than  black  with 
osmic  acid.  Pathologically  the  anisotropic  droplets  are  also  found 
especially  in  the  above-named  tissues,  but  also  in  tissues  the  site  of 
chronic  inflammation,  including  the  mucosa  of  the  gall  bladder  where 
they  may  be  of  importance  in  the  formation  of  cholesterol  concre- 
tions. They  are  also  found  in  the  alveolar  epithelium  in  pulmonary 
inflammation,  in  atheromatous  patches  in  arteries,  in  many  tumors, 
in  most  cells,"  including  even  the  adipose  tissues  themselves,-^  and 
occasionally  in  varied  pathological  tissues.'^  Perhaps  the  most  con- 
spicuous deposits  are  in  the  epithelium  of  the  "large  white  kidnej's," 
and  in  xanthomas.  In  Gaucher's  disease  there  is  also  a  remarkable 
lipoid  accumulation  in  the  foamy  phagocytic  cells. ^'^  According  to 
Munk"  true  lipoid  degeneration  always  means  a  serious  injury  to  the 
cell,  but  there  seem  to  be  many  exceptions  to  this.     Indeed,  according 

26  See  also  Verse,  Ziegler's  Beitr.,  1911  (52),  1. 
"  Ciaccio,  Cent.  f.  Path.,  1913  (24),  50. 

28  Cramer,  Jour.  Physiol.,  1917  (51),  xi. 

29  Pathological  decrease  in  lipoids  may  also  be  observed,  especially  in  the  ad- 
renal cortex,  usually  under  the  influence  of  toxic  agents;  e.  g.,  Hirsch  found  a 
marked  decrease  in  delirium  tremens  (Jour.  Amer.  Med.  Assoc,  1914  (63),  21S6). 

30  See  Wahl  and  Richardson,  Arch.  Int.  Med.,  1916  (17),  238. 

31  Virchow's  Arch.,  1908  (194),  527. 


408  RETROGRESSIVE  CHANGES 

to  Anitschkow  and  Chalatow  {loc.  cit).  the  feeding  of  foods  rich  in 
cholesterol  may  cause  the  appearance  in  the  liver  of  great  quantities 
of  anisotropic  droplets,  lipoid  deposits  in  the  aorta,  enlargement  of  the 
adrenal  cortex,  and  the  presence  in  practically  all  tissues  of  semifluid, 
doubly  refracting  crystalline  structures  (cholesterol  steatosis).^- 

In  cells  undergoing  autolysis  the  fat-like  "myehn"  droplets  which 
appear,  differ  from  the  above  in  not  being  anisotropic,  but  are  un- 
doubtedly closely  related  to  them  in  composition.  These  "myelin" 
droplets  are  also  found  in  cells  showing  cloudy  swelling,  presumably 
representing  cell  lipoids  set  free  through  changes  in  the  cell  proteins. 
They  are. characterized  by  staining  with  osmic  acid  but  not  by  sudan 
III,  which  shows  them  not  to  be  simple  fats  nor  yet  lipoids,  but  they 
are  undoubtedly  precursors  of  true  fatty  degeneration;^^  they  prob- 
ably consist  chiefly  of  lecithin,  with  more  or  less  free  fatt}'  acids  and 
relatively  little  cholesterol  (Aschoff). 

It  is  possible  to  distinguish  the  lipoids  of  cells,  whether  normal  or 
pathological,  from  the  neutral  fats  by  means  of  Ciaccio's  method.^* 
This  consists  in  a  preliminary  treatment  with  bichromate,  which  ren- 
ders the  lipoids  insoluble;  the  tissues  can  then  be  hardened  and  im- 
bedded by  the  usual  methods  which  remove  the  unchromated  fats, 
leaving  the  lipoids  stainable  by  sudan  III.  By  tliis  method  BelP° 
has  been  able  to  stain  the  lipoids  in  the  normal  kidney  and  other  tis- 
sues, in  sufficient  amount  to  account  for  all  the  so-called  "masked 
fat,"  which  thus  seems  to  be,  as  also  indicated  by  chemical  evidence, 
largely  lipoidal. 

Jastrowitz^^  has  studied  the  relation  of  lipoids  to  fats  in  the  fatty 
changes  produced  by  various  means,  and  finds  that  in  severe  fatty 
changes  with  much  transported  fats  there  may  be  little  change  in  the 
lipoids;  with  blood  poisons  which  cause  little  increase  in  total  fats, 
the  lipoid  content  of  both  blood  and  organs  may  be  high;  usually  the 
phosphatid  content  is  unchanged  or  slightly  increased,  but  it  may  be 
decreased.  The  proportion  of  cholesterol  to  neutral  fats  is  usually 
within  normal  limits  in  tissues  showing  fatty  changes.^'  The  mito- 
chondria seem  to  be  compounds  of  phospholipins  with  proteins,  and 
these  agglutinate  and  form  fathke  droplets  in  phosphorus  poison- 
ing.38  presumably  they  play  an  important  role  in  fatty  metamor- 
phosis. Cells  in  tissue  cultures,  however,  may  take  up  fat  droplets 
from  the  surrounding  medium  {i.  e.,  fatty  infiltration),  without  any 
association  with  or  changes  in  the  mito-chondria.^' 

"See  also  Anitschkow,  Deut.  med.  Woch.,  1913  (39),  741;  Wesselkin,  Vir- 
chow's  Arch.,  1913  (212),  225;  Rubinstein,  Compt.  Rend.  Soc.  Biol.,  1917  (80), 
191. 

33  Hess  and  Saxl,  Virchow's  Arch.,  1910  (202),  149. 

3^  Cent.  f.  Path.,  1909  (20),  771;  Arch.  f.  Zellf.,  1910  (5),  235. 

36  Jour.     Med.    Res.,     1911     (24),    539. 

38  Zeit.  exp.  Path.  u.  Ther.,  1914  (15),  116. 

3'  Czyhlarz  and  Fuchs,  Biochem.  Zeit.,  1914  (63),  131. 

38  Scott,  Amer.  .lour.  Anat.,  1916  (20),  237. 

39  M.  R.  Lewis,  Science,  1918  (48),  398. 


FATTY  METAMORPHOSIS  409 

Summary. — We  must  conclude,  tiierofore,  that  fatty  degeneration 
of  an  organ  means,  in  the  case  of  the  liver,  myocardium,  and  pan- 
creas, an  infiltration  of  neutral  fat  from  outside  into  cells  which  have 
been  degenerated  by  the  action  of  poisons  or  other  injurious  influ- 
ences, plus  a  certain  amount  of  apf)arent  increase  in  fat  because  of  the 
setting  free  of  previously  invisible  fats  and  lipoids  normally  present 
in  the  affected  cells.  In  the  kidney,  spleen,  and  muscles  an  increase 
of  fat  seldom  occurs  from  these  causes,  but  the  cells  may  show  a 
marked  fatty  metamorphosis  through  the  setting  free  of  the  invisible 
intracellular  fat  and  lipoids  by  autolytic  or  physico-chemical  changes. 
In  the  adrenal,  kidney,  and  often  in  other  tissues,  the  fatty  material 
present  in  the  cells  is  characterized  by  being  doubly  refractile,  and 
then  consists  chiefly  of  cholesterol  esters,  together  with  greater  or  less 
quantities  of  phosphatids,  fatty  acids,  soaps  and  neutral  fats. 

Pathogenesis  of  Fatty  Metamorphosis 

Nevertheless,  the  old  anatomical  distinction  of  infiltration  and  de- 
generation still  remains,  provided  we  do  not  hold  to  the  original  idea 
that  the  term  degeneration  implies  that  the  cell  protein  has  been  con- 
verted into  fat;  for  we  must  recognize  that  under  some  conditions  the 
cells  may  take  up  great  quantities  of  fat  without  suffering  any  appre- 
ciable degenerative  changes,  whereas  in  other  instances  the  appear- 
ance of  fat  is  associated  with  marked  and  complete  disintegration  of 
both  nucleus  and  cytoplasm.  Furthermore,  we  have  yet  to  explain 
why,  under  some  conditions,  the  fat  is  removed  from  the  fat  depots 
to  be  stored  up  in  the  liver  or  other  organs.  By  applying  the 
commonlj'  accepted  ideas  concerning  fat  metabolism,  a  satisfactory 
explanation  seems  to  be  possible.  Fat  is  always  utilized  and  trans- 
ported in  the  form  of  its  two  constituents,  fatty  acid  (or  soaps)  and 
glycerol,  which  are  diffusible  and  soluble.  It  enters  and  leaves  the 
cells  in  this  condition,  being  split  or  combined,  as  may  be  necessary  to 
produce  equihbrium,  by  the  action  of  lipase,  which  is  present  within 
the  cells  and  in  the  blood  and  lymph.  Under  normal  conditions  there 
is  little  free  visible  fat  in  the  cells  of  the  parenchymatous  organs, 
because  it  is  largely  used  up  through  oxidation  of  the  glycerol  and 
fatty  acids  by  the  action  of  the  intracellular  oxidases,  ^yhe^e  there 
is  abundant  lipase  and  but  little  oxidative  activity,  as  is  the  case  in 
the  areolar  fat  tissue,  fat  accumulates  in  large  amounts.  When,  for 
any  reason,  the  oxidative  power  of  the  parenchymatous  organs  is  re- 
duced, fat  accumulates  in  them  as  it  does  in  the  fat  depots  normally, 
and  we  have  an  excess  of  fat  in  the  parenchymatous  cells;  thus,  in 
pubnonary  tuberculosis,  severe  or  protracted  anemias,  etc.,  a  great 
accumulation  of  fat  occurs,  particularly  in  the  Kver,  where  normally 
active  oxidative  processes  continually  balance  the  action  of  the  abun- 
dant Hpase  of  the  liver-cells.  The  liver  being  normally  concerned  in 
the  preparation  of  fat  for  metabolism,  it  is  also  perfectly  possible  to 


410  RETROGRESSIVE  CHANGES 

have  an  accumulation  of  fat  in  the  normal  liver  merely  as  a  result  of 
increased  function,  and  hence  fatty  changes  may  be  purely  physio- 
logical in  this  organ. ■^'^ 

'  If  the  fat  accumulates  in  cells  that  are  structurally  normal  or 
nearly  so,  the  fat-droplets  fuse  together  under  the  pressure  of  the 
cytoplasm,  and  we  get  the  picture  of  a  typical  fatty  infiltration;  in- 
deed, the  only  tissues  in  which  we  get  this  typical  infiltration  are^the 
liver  and  the  fatty  areolar  tissue,  in  both  of  which  the  process  is  pre- 
sumably physiological  in  character  even  if  not  always  physiological 
in  degree.  If  the  cells  are  much  disintegrated  through  the  action  of 
the  poison, — e.  g.,  phosphorus,  bacterial  toxins,  etc., — ^the  accumulat- 
ing fat-droplets  are  not  crowded  into  one  large  droplet,  but  He  free 
in  the  granular  debris  of  the  disintegrating  cell,  constituting  the  typi- 
cal appearance  of  fatty  degeneration.  Fatty  degeneration  is  usually 
brought  about  by  poisons,  while  abnormal  fatty  infiltration  depends 
usually  upon  decreased  oxidation,  due  to  lack  of  either  oxygen  or 
hemoglobin  in  the  blood.  If  the  anemia  is  extreme,  however,  the  cells 
degenerate,  and  then  we  find  a  true  fatty  degeneration  caused  by  lack 
of  oxygen. "^^  Thus,  in  an  anemic  infarct  fat  accumulates  about  the 
periphery  of  the  dead  area,^^  probably  because  fatty  acids  and  glycerol 
diffuse  in  slowly  from  the  surrounding  parts  where  circulation  still 
goes  on,  and  are  built  up  into  fat  by  the  cell  lipase,  for  in  anemic  areas 
the  intracellular  oxidases  cannot  destroy  these  substances  as  they  nor- 
mally do,  because  of  lack  of  oxygen.  The  accumulation  of  fat  in  dead 
areas  depends,  therefore,  on  the  fact  that  the  constituents  of  fat  can 
diffuse  into  the  dead  tissue,  whereas  the  oxygen,  being  held  in  the  cor- 
puscles, cannot  enter  the  anemic  area.^^  It  is  also  possible  that  where 
fat  is  set  free  by  autolysis  of  dead  tissue,  or  when  for  any  cause  free 
fat  or  lipoid  material  is  present  in  the  vicinity  of  living  cells,  it  may 
be  phagocyted  or  in  some  way  infiltrate  the  cells,  causing  a  fatty  meta- 
morphosis by  absorption  (Dietrich). 

It  is  to  be  supposed  that  poisons  also  cause  fatty  degeneration  in 
a  similar  way — -by  interfering  with  oxidation.  We  have  much  evi- 
dence that  in  phosphorus,  chloroform,  and  other  poisoning  associated 
with  fatty  degeneration  of  the  liver,  oxidation  is  impaired."*^  If  we 
imagine  for  a  moment,  a  cell  in  which  oxidation  is  checked  by  any 
means,  we  shall  have  in  this  cell  the  lipase  and  the  proteol3^tic 
enzymes  not  balanced,  as  they  normally  are  by  the  action  of  the  oxi- 
dases, and  hence  the  processes  of  cell  autolysis  and  of  the  accumula- 

"  See  Coope  and  Mottram,  Jour,  of  Physiol.,  1914  (49),  23;  Helly,  Beitr.  path. 
Anat.,  1914  (60),  1. 

^'  Mohr  (Zcit.  exp.  Path.,  190G  (2),  434),  denies  that  oxidation  is  decreased  in 
anemia;  and  in  a  man  with  but  about  half  the  normal  lung  area  the  metabolism 
was  not  found  iiltored  to  any  extent  by  Carpenter  and  Benedict,  Amer.  Jour. 
Physiol.,  1909  (23),  412. 

«  Fisciiler,  Cent.  f.  Path.,  1902  (13),  417. 

"  See  Griesser,  Ziegler's  Beitr.,  1911  (51),  115. 

"  See  Welsch,  Arch.  int.  de  pharm.  et  therap.,  1905  (14),  211. 


FATTY  METAMORPHOSIS  411 

tion  of  fat  by  the  lipase  will  go  on  uncontrolled.  The  result  will  be 
a  disintegrated  cell  containing  many  fat-droplets,  i.  e.,  fatty  degen- 
eration.^^ In  cloudy  swelling  there  also  appear  droplets  stained  with 
osmic  acid  but  not  by  sudan  111,  which  Hess  and  Sa.xl"'  have  shown 
to  result  from  intravitam  cell  autolysis,  and  to  be  a  precursor  of  true 
fatty  degeneration. 

Work  with  cells  in  tissue  cultures  indicates  that  fatty  changes  of 
all  types  may  occur  independently  of  the  circulation.  Lamberf^ 
states  that  the  amount  of  fat  in  the  culture  cells  is  roughly  propor- 
tional to  the  amount  in  the  culture  medium,  and  cells  rich  in  fat  may 
move  actively  and  undergo  normal  mitosis.  Lewis,  however,  observed 
fatty  changes  in  cells  growing  in  fat-free  media,  and  made  the  espe- 
ciall}''  interesting  observation  that  cells  grown  in  2.5-3  per  cent,  alco- 
hol will  show  a  rich  fat  accumulation.  Also,  an  accumulation  of  fats 
and  lipoids  in  cells  grown  in  the  presence  of  such  steatogenetic  poi- 
sons as  phosphorus  and  Oleu7n  pulegii  has  been  observed  by  others, "'^ 
which  indicates  that  free  cells  behave  the  same  under  the  influence 
of  such  poisons  as  the  cells  of  the  fixed  tissues. 

The  process  of  unmasking  the  masked  fats  is  explained  by  M.  H. 
Fischer^''  on  a  physical  basis,  as  follows:  The  fats  of  the  cells  are 
distributed  as  an  emulsion  in  a  hydration  compound  of  water  with 
hydrophilic  colloids,  notably  proteins  and  soaps.  Such  an  emulsion 
breaks  down  whenever  the  hydrophilic  colloid  is  either  dehydrated  or 
diluted  beyond  certain  ranges.  As  the  usual  conditions  that  cause 
fatty  degeneration,  such  as  poisoning  with  phosphorus,  arsenic,  etc., 
or  local  circulatory  disturbances  with  local  acidosis,  all  tend  to  de- 
hydrate some  of  the  cell  colloids  and  to  dilute  others,  it  would  seem 
probable  that  the  appearance  of  the  fat  droplets  in  the  cells  is  the 
result  of  such  changes  in  the  colloids  that  previously  held  them  in  an 
emulsion  too  fine  to  exhibit  readily  visible  fat  particles.  The  relation 
of  cloudy  swelling  to  fatty  degeneration  is  readily  explained  on  this 

^^  Interference  with  oxidation  does  not  necessarily  irnply  destruction  of  the 
oxidases.  As  yet  we  know  practically  nothing  concerning  the  oxidases  of  the 
cells  in  disease,  and  the  above  hypothesis  has  yet  to  be  demonstrated.  Duccheschi 
and  Aluiagia  (Arch.  Ital.  Biol.,  1903  (39),  29)  found  the  normal  amount  of  lipase 
in  phosphorus-livers,  but  also  observed  no  decrease  in  ability  to  oxidize  salicylic 
aldehyde,  which,  however,  does  not  prove  a  normal  power  to  oxidize  fats.  Gierke's 
observation  (Ziegler's  Beitr.,  1905  (37),  502)  that  glycogen  and  fat  accumulate 
under  identical  conditions  might  be  cited  as  indicating  decreased  oxidative  power. 
Wells  (Jour.  Exper.  Med.,  1910  (12),  607)  found  that  the  power  of  liver  tissue  to 
oxidize  purines  was  not  decreased  by  the  maximum  degree  of  fatty  degeneration, 
but  Waldvogel  (Deut.  Arch.  klin.  Med.,  1907  (89),  342)  found  that  obese  persons 
can  burn  fatty  acids  which  arise  in  metabolism  less  readily  than  normal;  and  Quinan 
(.Jour.  Med.  Res.,  1915  (32),  73)  found  the  ester-splitting  lipolytic  enzymes  of  the 
liver  much  reduced  in  the  liver  of  chloroform  necrosis,  but  the  relation  of  these 
esterases  to  true  lipases  is  not  known. 

^«  Virchow's  Arch.,  1910  (202),  149. 

*'  Trans.  Assoc.  Amer.  Phys.,  1913  (9),  93;  Jour.  Exp.  Med.,  1914  (19),  398. 

"  Krontowski  and  Poteff,  Beitr.  path.  Anat.,  1914  (58),  407. 

^^  Fischer  and  Hooker,  Science,  1910  (43),  468;  Fischer,  Fats  and  Fatty  Degen- 
eration, Wiley,  New  York,  1917. 


412  RETROGRESSIVE  CHANGES 

basis,  as  follows:  When  a  local  acid  intoxication  of  a  cell  occurs, 
some  of  the  proteins  will  swell  and  others  will  precipitate,  resulting 
respectively  in  the  swelling  and  cloudiness  of  the  cells  characteristic 
of  cloudy  swelhng;  but  at  the  same  time  the  emulsifying  capacity  of 
these  proteins  will  be  impaired,  permitting  the  coalescence  of  the  fat 
droplets  and  the  resulting  picture  will  be  that  of  fatty  degeneration. 
Summary .^ — Fatty  metamorphosis  involves  changes  of  two  kinds. 
First,  infiltration  of  fat,  which  occurs  when  the  oxidative  power  of 
the  cells  is  decreased,  so  that  fat  is  not  destroyed,  but  is  accumulated 
from  the  blood  under  the  influence  of  the  hpase  of  the  cells;  if  there 
is  not  any  serious  injury  to  the  cells,  the  histological  changes  consist 
in  the  accumulation  of  one  or  a  few  large  droplets  of  fat  in  each  cell, 
constituting  the  condition  known  anatomically  as  ''fatty  infiltration." 
This  occurs,  pathologically,  chiefly  in  the  Hver.  If  at  the  same  time 
the  cytoplasm  is  disintegrated  through  autolylic  changes,  the  fat- 
droplets  do  not  fuse,  but  remain  as  small,  more  or  less  discrete,  fat 
granules  among  the  granules  of  cell  debris,  constituting  the  micro- 
scopic picture  of  "fatty  degeneration";  this  condition  occurs  particu- 
larly in  the  heart  and  hver. 

Second,  each  cell  contains  a  large  amount  of  fat  and  hpoids  (5-25 
per  cent,  of  its  dry  weight),  which  is  so  combined  that  it  cannot  be 
detected  microscopically;  this  may  be  hberated  during  the  autolytic 
processes  and  colloidal  changes  of  cell  disintegration  and  become 
visible,  constituting  a  macroscopical  and  microscopical  degeneration, 
but  without  any  actual  increase  in  fat — this  condition  occurs  particu- 
larly in  the  kidney  and  nervous  system.  Third,  a  combination  of 
both  of  the  above  processes — infiltration  of  fat  and  liberation  of 
masked  intracellular  fat — may  occur  simultaneously  in  an  organ. *^ 
Fourth,  in  certain  cells,  especially  in  the  kidney,  adrenal,  ovary  and 
some  tumors,  there  may  be  a  great  increase  in  the  lipoids  of  the  cell, 
''lipoidal  degeneration,"  and  especially  of  cholesterol  esters  and  free 
cholesterol,  part  of  which  is  infiltrated  and  part  set  free  from  com- 
bination in  the  cytoplasm. 

PROCESSES  RELATED  TO  FATTY  METAMORPHOSIS 
ADIPOCERE 

This  apparent  transformation  of  the  substance  of  dead  bodies  into 
a  wax-like  material  was  for  a  long  time  looked  upon  as  evidence  of  a 
transformation  of  protein  into  fat,  but  in  the  light  of  more  recent  in- 
vestigations this  view  can  hardly  be  held.  Adipocere  is  the  product 
of  a  process  that  occurs  particularly  in  bodies  buried  in  very  wet 

^'  The  above  conception  of  the  processes  involved  in  fatty  metamorphosis  is 
more  fully  discussed  by  the  writer  in  other  publications  (Jour.  Amer.  Med.  .\ssoc., 
1902  (38),  220;  ibid.,  1906  (46;,  341).  Ribbert  (Deut.  med.  Woch.  1903  (29), 
793)  has  also  advanced  a  similar  explanation  for  the  morphological  differences 
between  fatty  "degeneration"  and  "infiltration,"  i.  e.,  that  the  degenerative 
changes  are  independent  of  fatty  accumulation. 


ADIPOCERE 


413 


places  or  lying  in  water,  and  results  in  an  apparent  replacement  of 
the  muscles  and  other  soft  parts  (but  not  the  glandular  organs)  by  a 
mass  consisting  of  a  mixture  of  fatty  acids  in  crystaUine  and  amor- 
phous form,  and  soaps,  particularly  ammonium,  magnesium,  and 
calcium  salts  of  palmitic  and  stearic  acid  (the  oleic  acid  largely  disap- 
pearing during  the  process).  Analysis  of  samples  of  adipocere  by 
Ruttan^-  gave  the  following  figures: 

Composition  of  Human  and  Pig's  Adipocere 


I 

II 

III 

Pigs 

Human 

Human 

(mature) 

hard 

soft 

94.1 

82.9 

75.8 

0.8436 

0.8397 

0  8410 

1.436 

1.437 

1.439 

60-63° 

52-54° 

50-51° 

201.7 

207.3 

203.8 

207.0 

211.0 

212.2 

6.04 

9.65 

12.52 

34.75 

11.8 

271.0 

266.0 

264  0 

70.82 

71.78 

59.2 

5.24 

8.87 

11.6 

14.80 

8.24 

7.8 

1.21 

0.91 

0.90 

0.16 

0.15 

0.83 

0.87 

0.69 

0.75 

4.41 

6.76 

12.3 

0.665 

1.93 

4.14 

0.035 

0.054 

0.574 

i.99 

2  25 

Ether  soluble,  per  cent 

Specific  gravity  at  100°  C. . . 

Refractive  index  65°  C 

Melting-  point 

Acid  value 

Saponification  value 

Iodine  value 

Acetyl  value 

Mean  molecular  weight 

Saturated  fat  acids,  per  cent 

Unsaturated  fat  acids 

Hydroxy  fat  acids 

Stearin  and  palmitin 

Olein 

Unsaponified  matter 

Calcium  soaps 

Protein 

Ammonia 

Ash 


Ammonium  and  other  soluble  soaps  were  absent.  The  hydroxy- 
stearic  acids,  w^hich  are  so  characteristic  of  adipocere,  are  formed  from 
the  oleic  acid  of  the  original  triolein.  Cholesterol  has  also  been  found 
in  adipocere.  ^^ 

The  resulting  material  is  absolutely  resistant  to  putrefaction,  and 
hence  remains  intact  for  many  years.  This  replacement  of  the  soft 
parts  is,  however,  only  apparent,  for  the  total  weight  of  a  body  in 
this  condition  is  much  Hghter  than  that  of  the  original  body;  indeed, 
one  is  always  surprised  at  the  hght  weight  on  Hfting  such  a  specimen. 
Adipocere  occurs  almost  exclusively  in  fat  bodies,  and  it  seems  probable 
that  all  the  soaps  and  fatty  acids  found  are  formed  from  the  original 
fats  of  the  corpse.^^  These  gradually  flow  into  the  places  left  by  the 
disintegrating  muscle,  etc.,  a  process  that  occurs  readily  in  cadavers, 
according  to  Zillner;"^  or  the  infiltration  may  be  accomplished  through 

"Jour.  Biol.  Chem.,  1917  (29),  319;  Trans.  Rov.  Soc.  Can.,  1916  (10),  169. 
*'  Van  Itallie  and  Steenhauer,  Pharm.  Weekblad,  1917  (54),  121. 
"  Fatty  changes  in  the  viscera  may  favor  their  transformation  into  adipocere 
(Muller,  Vierteljahrs.  gericht.  Med.,  1915  (50),  251). 
"  Vierteljahrsch.  f.  gericht.  Med.,  1885  (42),  1. 


414  RETROGRESSIVE  CHANGES 

diffusion  of  the  ammonium  soaps  formed  during  the  decomposition. 
As  the  subcutaneous  fat  is  hardened  by  the  formation  of  soaps,  and  the 
bones  remain  to  hold  the  parts  in  position,  the  general  form  of  the 
body  is  preserved,  creating  the  impression  that  its  entire  substance  has 
been  converted  into  adipocere,  when  the  total  mass  may  actually 
weigh  but  twenty  pounds  or  so,  and,  according  to  Zillner's  estimate, 
not  more  than  one-tenth  of  the  muscle  substance  is  replaced  by  adi- 
pocere. This  false  impression  is  probably  responsible  for  much  of  the 
mistaken  idea  concerning  the  conversion  of  tissue  proteins  into  fatty 
acids.  Thus,  Schmidt^''  found  that  in  early  Egyptian  mummies  60 
per  cent,  of  the  weight  of  the  lungs  and  30  per  cent,  of  the  spleen  con- 
sisted of  fatty  acids,  and  fell  into  the  usual  error  of  considering  this 
conclusive  evidence  of  transformation  of  proteins  into  fat. 

Numerous  attempts  have  been  made  to  prove  that  muscle  could 
be  thus  converted  into  fatty  acids  and  soaps,  but  although  success  has 
been  claimed  by  a  few,  the  results  are  not  entirely  convincing.  ^^ 
Bacteria  can  convert  proteins  into  fats,  beyond  a  doubt,  and  they 
may  do  so  to  some  slight  extent  in  adipocere  formation,  but  probably 
this  factor  is  not  important. 

In  the  light  of  our  present  conception  of  fat  metabohsm  it  is  prob- 
able that  the  process  of  adipocere  formation  occurs  as  follows:  The 
fatty  acids  of  the  fat  tissue  are  combined  by  the  ammonia  formed 
during  putrefaction,  removing  these  fatty  acids  from  the  normal 
balance  of  fat  and  fatty  acids  in  the  fat  tissue;  as  a  result,  the  lipase 
of  the  fat  tissue  continues  to  split  the  fat,  and  more  fatty  acids  are 
produced,  which  likewise  go  to  form  soaps.  This  continues  until 
practically  all  the  neutral  fat  has  been  decomposed,  the  glycerol  dif- 
fusing rapidly  away.  The  soluble  soaps,  which  the  bacteria  do  not 
attack,  diffuse  into  the  softened  muscle  tissue,  which  they  gradually 
replace  in  part.  In  the  meantime,  from  the  more  soluble  ammonium 
soaps,  calcium  and  magnesium  soaps  are  being  slowly  formed,  accord- 
ing to  the  usual  rule  of  double  decomposition  (that  the  least  soluble 
salt  will  be  formed  under  such  conditions) ;  or  else,  if  an  acid  reaction 
develops,  free  fatty  acids  are  precipitated.  The  oleic  acid  seems  to  be 
converted  into  the  higher  fatty  acids  (Salkowski).-""*  It  is  also  possible 
that  the  saponification  is  due  to  the  gradual  action  of  the  alkaline 
fluids  produced  in  decomposition  of  the  tissues,  or  to  the  alkalinity 
of  the  water  in  which  the  body  lies.  Possibly  bacteria  may  be  re- 
sponsible for  this  decomposition  of  the  fats  rather  than  the  body 
lipase,  for  Eijkman^^  has  observed  that  certain  bacteria  growing  in 
fat-containing  agar  produce  calcium,  ammonium,  and  sodium  soa[is, 
simulating  adipocere. ^'^ 

"  Zeit.  allg.  Physiol.,  1907  (7),  3G9. 

"  See  Rosenfeld,  Ergcb.  dor.  Physiol.,  Abt.  1,  1902  (1),  659. 
"  Festschr.  f.  Virchow,  1S91,  p.  23;  corroliorated  by  Schiitze. 
"Cent.  f.  Bakt.,  1901  (29),  847. 

«»See  also  Ccvidalli,  \'it>rteljahrschr.  froiifhtl.  Mod.,  190G  (32),  219;  and 
Schiitze,  Arch.  Ilyg.,  1912  (70),  110. 


LIPEMIA  415 

Zillner**  gives  the  following  scheme  of  the  changes  that  take  place 
in  a  cadavrr  undergoing  aflipocorc  formation:  (1)  Migration  of  fluid 
contents  of  the  body  (imbibition  of  blood  and  transudation) — one  to 
four  weeks.  (2)  Decomposition  of  superficial  epidermis,  then  of 
corium — ^first  two  months.  (3)  Decomposition  of  muscle  and  gland 
parenchyma,  until  only  the  inorganic  part  of  the  bones  and  the  con- 
nective and  elastic  tissues  remain — three  to  twelve  months.  (4) 
Migration  of  neutral  fat,  crystallization  and  partial  saponification  of 
the  higher  fatty  acids  in  the  panniculus;  transformation  of  the  blood 
pigment   into  crystalline  form — four  to   twelve  or  more    months. ^^ 

LiPEMIA 

Normally  the  blood  contains  a  considerable  amount  of  fats  and 
lipoids,  varying  somewhat,  but  not  greatly,  with  the  diet.  The  older 
literature  gave  figures  varying  widely,  but  analyses  by  more  modern 
methods"-  give  figures  for  the  ether-soluble  constituents  of  the  normal 
plasma  (before  breakfast)  ranging  ordinarily  from  0.57  to  0.82  per 
cent.,  of  which  cholesterol  and  phosphatid"''  are  about  equal  (0.2  to  0.3 
per  cent.)  with  very  httle  neutral  fat  (0.1  to  0.2  per  cent.).  In  various 
diseases,  exclusive  of  diabetes,  the  total  lipin  content  was  found  by 
Bloor  to  be  about  normal,  but  the  proportion  of  the  different  lipins 
varied  somewhat.  After  taking  fat-rich  food,  however,  there  may  be 
a  considerable  excess  of  the  food  fats  in  the  serum,  and  it  is,  there- 
fore, extremely  difficult  to  say  just  when  the  amount  of  fat  in  the  blood 
is  large  enough  to  be  considered  as  a  lipemia,  especially  since  after  every 
fatty  meal  there  is  enough  fat  in  the  blood  to  make  it  turbid.'"'^"  B. 
Fisher"*  states  that  we  may  speak  of  a  pathological  lipemia  when  we 
have  a  distinctly  cloudy  blood  or  serum,  which  is  clarified  by  shaking 
with  ether  through  the  dissolving  out  of  fat  which  can  then  be  sepa- 
rated from  the  ether.  We  may,  however,  sometimes  find  turbid 
plasma  with  normal  hpin  content  and  clear  plasma  with  hyperlipemia 
(Gray).  Earlier  writers  described,  incorrectly,  lipemia  in  many  con- 
ditions, but  recent  writers  mention  it  chiefly  as  occurring  in  alcoholism"^ 
and  diabetes.  By  far  the  greatest  amounts  of  fat  are  observed  in  the 
latter  condition,  and  diabetic  lipemia  is  always  accompanied  by  an 
acidosis,  although   acidosis   often   occurs   without  lipemia.     Experi- 

^*  Sclerema  neonatorum  is  caused  by  hardening  of  the  subcutaneous  fat,  perhaps 
because  of  a  low  proportion  of  oleic  acid.  (Beyer,  Verh.  Deut.  Path.  Gesell.,  1908 
(12),  .305.)  C.  S.  Smith,  however,  found  normal  oleic  acid  but  a  high  figure  for 
free  fatty  acids.  Others  have  described  high  melting  points  for  the  fat,  believing 
the  condition  to  be  merely  an  exaggeration  of  the  normally  high  proportion  of 
palmitic  and  stearic  acids  of  infant  fat  tissues  (Smith,  Jour.  Cut.  Dis.,  1918  (36), 
436). 

«2  Bloor,  Jour.  Biol.  Chem.,  1916  (25),  577. 

^^  Concerning  blood  lecithin  see  Feigl,  Biochem.  Zeit.,  1918  (90),  361. 

®^"  Neisser  and  Brauning,  Zeit.  exp.  Bath.  u.  Ther.,  1907  (4),  747. 

"  Virchow's  Arch.,  1903  (172),  30.     R6sum6  and  complete  literature. 

^^  Also  occurs  in  'experimental  alcoholism  (Feigl,  Biochem.  Zeit.,  1918  (92), 
282;  Bang,  ibid.,  (90),  383). 


416  RETROGRESSIVE  CHANGES 

mental  pancreatic  diabetes  may  be  accompanied  by  lipemia.^^  In- 
creases in  the  blood  lipoids,  not  usually  of  sufficient  magnitude  to  cause 
a  distinct  lipemia,  may  be  found  in  nephritis  (Bloor),  cirrhosis,  tabes 
and  paralysis  (Feigl).^^ 

Neisser  and  Derlin^^  found  19.7  per  cent,  of  fat  in  the  blood  of  a 
patient  with  diabetic  coma  (after  death  24.4  per  cent,  was  found) 
whose  urine  contained  0.8  per  cent,  of  fat,  and  through  analysis  of 
this  and  other  material  came  to  the  conclusion  that  the  fat  comes 
directly  from  the  chyle;  i.  e.,  that  it  is  food  fat,  not  body  fat.  Fischer 
found  an  average  of  18.129  per  cent,  in  his  case,  including  at  least 
0.478  per  cent,  of  cholesterol,  with  no  lipuria  and  very  small  amounts 
of  fatty  acids;  of  the  fat,  about  67.5  per  cent,  was  olein.  Ringer®^ 
has  found  14.4  per  cent,  of  lipins,  including  2.14  per  cent,  of  cholesterol. 
As  high  as  27  per  cent,  of  fat  has  been  found  in  the  blood.''"  In  many 
cases  the  increase  is  chiefly  in  the  lipoids,  liyoidemia,"^^  and  in  acidosis 
there  is  said  to  be  an  especial  increase  in  cholesterol  i^Adler).''^ 

Study  of  a  large  number  of  diabetic  bloods  by  Gray'^^  gave  the 
following  results:  Normal  lipin  values  are  seldom  found,  the  most 
marked  increases  being  in  the  total  glycerides,  next  the  total  fatty 
acids,  then  the  cholesterol,  and  least  the  phospholipins.  Increase  of 
both  cholesterol  and  glycerides  seems  to  be  pathognomonic  of  chronic 
diabetic  lipemia,  as  in  alimentary  hpemia  the  increase  is  in  the  fatty 
acids.  The  greater  the  duration  of  the  diabetes  the  lower  the  lipins, 
and  high  figures  give  a  bad  prognosis,  being  usually  associated  with 
acidosis.  Hyperglycemia  and  hyperlipemia  do  not  run  parallel.^* 
In  general,  the  amount  of  blood  lipins  increases  with  the  severity  of  the 
disease, ^^  the  averages  in  a  large  series  of  analyses  being  as  follows: 
normal,  0.59  per  cent.;  mild  diabetes,  0.83;  moderate,  0.91;  severe,  1.41. 
The  changes  concern  chiefly  the  plasma.  Coexistent  nephritis  does 
not  modify  the  blood  hpin  figures.  When  the  lipemia  is  accompanied 
by  icterus  the  fats  may  clear  up  and  a  clear  serum  is  present,  despite 
a  high  fat  content. ^"^ 

It  is  an  important  question  whether,  with  high  quantities  of  fat 
in  the  blood,  fat  embolism  may  result,  for  it  is  possible  that  at  least 
some  of  the  cases  of  diabetic  coma  are  due  to  such  fat  embolism  in 
the  cerebral  vessels.  Ebstein^^  considers  this  a  possible,  but  not  a 
common,  occurrence,  because  the  droplets  are  too  small  to  cause  oc- 

"  Seo,  Arch.  exp.  Path.  u.  Pharm.,  1909  (Gl),  1. 

"  Biochem.  Zeit.,  1918  (88),  53;  (90),  1. 

«»  Zeit.  klin.  Med.,  1904  (51),  428. 

"o  Proc.  Soc.  Exp.  Biol.  Med.,  1917  (15),  40. 

^»  Frugoni  and  Marchetti,  Bed.  klin.  Woch.,  1908  (45),  1844. 

^1  See  Weil,  Munch,  med.  Woch.,  1912  (59),  2096. 

"  Berl.  klin.  Woch.,  1910  (47),  1323. 

"  Boston  Med.  Surg.  Jour.,  1917  (178),  16. 

'*  Corroborated  by  Bang,  Biochem.  Zeit.,  1919  (94),  359. 

"  See  Jour.  Amer.  Med.  Assoc,  1917  (69),  375. 

'8  Feigl  and  Querner,  Zeit.  exp.  Med.,  1919  (9),  153. 

"  Virchow's  Arch.,  1899  (155),  571. 


LIPEMIA  417 

elusion  of  the  vessels  unless  they  conibine  to  form  large  droplets. 
Fischer  doubts  if  the  droplets  ever  fuse  together  enough  to  cause  em- 
bolism, supporting  his  contention  both  by  experiments  and  clinical 
records,  but  cases  have  been  reported  as  fat  emboUsm  from  diabetic 
lipemia.'^^ 

The  cause  of  lipemia  has  not  yet  been  satisfactorily  determined. 
In  alcohohsm  it  is  commonly  ascribed  to  a  failure  to  burn  fat,  because 
of  the  presence  of  the  more  readily  oxidized  alcohol,  and  the  common 
coexistence  of  diabetes  and  hpemia  suggests  for  both  a  common  cause; 
i.  €.,  lack  of  oxidation  of  fat  and  sugar.  In  corroboration  may  be 
cited  the  occurrence  of  lipemia  in  other  conditions  associated  with 
defective  oxidation;  i.  e.,  pneumonia,  anemia,^'-*  phosphorus-poisoning. 
As  we  are  still  unfamiliar  with  the  essential  factors  and  steps  in  the 
oxidation  of  fat,  it  would  be  mere  speculation  to  attempt  to  explain 
further  the  reason  for  the  failure  of  destruction  of  the  fat.  The  origin 
of  the  fat  in  hpemia  is  likewise  undetermined.  Ebstein  considers 
that  it  arises  partly  from  the  food,  partly  from  fatty  degeneration  of 
the  cells  of  the  blood,  the  vessel-walls,  and  the  viscera.  Neisser  and 
Derlin  consider  it  as  merely  food  fat  coming  from  the  chyle  and 
accumulated  in  the  blood.  Fischer  beheves  that  it  is  largely  derived 
from  the  fat  depots,  and  that  because  of  loss  of  the  Hpolytic  power 
of  the  blood  it  cannot  be  rendered  diffusible,  and  hence  it  cannot  enter 
the  tissues  where  it  is  normally  consumed.  Sakai^"  also  found  a  low 
lipase  content  in  the  blood  and  suggests  that  fat  entering  the  blood  is 
unable  to  leave  it  because  of  defective  hpolysis.  Klemperer  and  Um- 
ber hold  that  it  comes  from  disintegration  of  tissue  cells,  but  are 
unable  to  determine  the  cells  concerned.  Ervin^^  attributes  diabetic 
hpemia  to  the  glycogen  deficiency  of  the  cells,  assuming  that  glycogen 
acts  as  a  protective  colloid  which  holds  the  intracellular  fats  in 
emulsion. 

Bloor's  studies^-  support  strongly  the  view  that  the  fats  come 
from  the  food,  for  he  found  hpemia  only  in  diabetics  receiving  fat  in 
their  food,  and  under  fasting  an  existing  hpemia  disappears.  Choles- 
terol increases  parallel  with  the  fat,  while  lecithin  is  relatively  little 
increased.  Verse^^  says  that  a  lasting  lipemia  can  be  produced 
by  feeding  rabbits  mixed  cholesterol  and  oil,  but  not  with  either  of 
these  alone.  In  severe  diabetes  without  hpemia  the  hpins  are  all  much 
increased  in  the  plasma,  but  with  the  relative  proportions  about  as  in 
normal  individuals,  although  with  a  tendency  for  the  fats  to  accumu- 
late in  excess.  The  facts  that  fat  oxidation  depends  upon  carbohy- 
drate oxidation,  and  also  that  in  diabetics  excessive  fat  feeding  is 

"8  Hedren,  Svenska  Liik.  Handl.,  1916  (42),  933. 

"  See  Boggs  and  Morris  (Jour.  Exper.  Med.,  1909  (11),  553),  who  produced 
lipemia  bv  repeatedly  bleeding  rabbits. 
8"  Biochem.  Zeit.,  1914  (62),  387. 
"  Jour.  Lab.  Clin.  Med.,  1919  (5),  146. 
"  Jour.  Biol.  Chem.,  1916  (26),  417;  1917  (31),  575. 
83  Miinch  med.  Woch.,  1916  (63),  1074. 

27 


418  BETROGRESSIVE  CHANGES 

usual,  are  probably  significant  in  the  causation  of  diabetic  lipemia. 
(See  also  cholesterolemia.) 

Pathological  Occurrence  of  Fatty  Acids 

Fatty  acids  occasionally  occur  free  in  pathological  processes.  The 
best  example  of  this  is  fat  necrosis  (q.  v.).,  where  crystals  of  fatty  acids 
appear  in  the  necrotic  fat-cells,  arising  through  splitting  of  fat.  and 
later  becoming  combined  with  calcium  from  the  blood.  Similar 
crystals,  consisting  of  a  mixture  of  palmitic  and  stearic  acids,  fre- 
quently called  margarin  or  inargaric  acid  crystals,  may  be  found  in 
decomposed  pus,  in  sputum  from  bronchiectatic  cavities  and  from 
gangrene  of  the  lungs,  in  gangrenous  tissue,  and  in  atheromatous  areas. 
According  to  Schwartz  and  Kayser^'*  the  free  fatty  acids,  at  least  in 
pulmonary  gangrene,  arise  from  Hpolysis  by  bacterial  action  rather 
than  by  the  hpase  of  the  tissues.  Eichhorst  found  crystals  of  fatty 
acids  in  the  neighborhood  of  acute  patches  of  sclerosis  in  the  central 
nervous  system  in  multiple  sclerosis,  and  McCarthy*^  found  them 
in  a  spinal  cord  undergoing  secondary  degeneration  from  compression. 
Whipple^^  describes  a  case  with  deposits  of  fatty  acids  and  neutral 
fat  in  the  wall  of  the  intestine  and  the  mesenteric  glands,  while  soaps 
and  fatty  acids  are  said  to  be  present  in  excess  in  chronic  appendicitis.^^ 
Soaps  and  fatty  acids,  especially  oleic  acid  and  oleates,  are  highly 
toxic,  and  their  profound  hemolytic  power  has  been  thought  of  im- 
portance in  pathological  conditions,  especially  bothriocephalus  anemia.  ^^ 
(See  Hemolysins,  Chap,  ix.)  The  fatal  dose  of  sodium  oleate  for 
rabbits  is  0.15  gm.  per  kilo  (Leathes).  The  salts  of  higher  fatty  acids 
above  capric  are  hemolytic,  while  those  from  caproic  down  are  not, 
nonoic  acid  salts  being  the  turning  point  (Shimazono).^^  The  toxicity 
of  soaps  may  be  related  to  their  marked  power  to  inhibit  proteolytic 
enzymes.^" 

The  fatty  acids  may  be  stained  green  by  copper  acetate,  according 
to  Benda's  method,  and  if  then  treated  with  hematoxylin,  they  turn 
black. ^^  With  Nile  blue  sulphate  they  stain  blue,  forming  a  blue 
salt,  while  the  neutral  fats  are  stained  red  by  the  oxazone  base  (J.  L. 
Smith).  Fischler  and  Gross^-  state  that  fatty  acids  are  present  in 
atheromatous  areas  and  about  the  margin  of  anemic  infarcts,  but 
are  not  recognizable  by  this  method  in  such  fatty  degenerations  as 
pneumonic  exudates,  caseation,  etc.     Klotz^^  considers  that  calcium 

«<  Zcit.  klin.  Med.,  1905  (56),  111. 

"Univ.  of  Penn.  Med.  Bull.,  1903  (IG),  141. 

««  liull.  Johns  Hopkins  Hosp.,  1907  (IS),  382. 

"Anthony,  Jour.  Med.  Res.,  1911  (20),  359. 

'*  Faust,  Suppl.  Bd.,  Schiuiedebcrp's  Arch.,  1908,  p.  171. 

89  Z.  Immunitat.,  Ilef.,  1911  (4),  650. 

"".lobling  and  Petersen,  Jour.  Exp.  IMod.,  1914  (19),  251. 

«'  Fischler,  Cent.  f.  Path.,  1904  (15),  913. 

82  Ziegler's  Beitr.,  1905  (7th  suppl.),  343. 

"  Jour.  E.\p.  Med.,  1905  (7),  633. 


rATIIOLoaiCAL  OCCURRENCE  OF  CHOLESTEROL  419 

soaps  are  forinod  as  the  fiist  step  in  pathological  calcification,  accord- 
ing to  microchciuical  evidence;  but  a  chemical  investigation  of  the 
same  question  did  not  give  the  writer  positive  results.'-"  In  fatty 
cells,  especially  in  the  liver,  crystals  are  often  found  and  interpreted 
as  fatty  acids,  which  are  really  crystals  of  neutral  fats."^ 

Pathological  Occurrence  of  Cholesterol" 

Cholesterol  in  crj^stals  is  found  under  somewhat  the  same  conditions 
as  the  fatty  acids,  and  although  cholesterol  is  not  a  fat,  but  an  alco- 
hol, its  ph3^sical  properties  are  so  similar  that  it  may  be  considered 
in  this  place.  (See  "Gall-stones,"  Chap,  xvii,  for  further  discussion.) 
The  characteristic  large  flat  plates  of  cholesterol  may  be  found  in  any 
tissue  in  which  cells  are  undergoing  slow  destruction,  and  where  absorp- 
tion is  Y>oor.  Therefore,  they  are  found  frequently  in  atheromatous 
patches  in  the  blood-vessels,  encapsulated  caseous  areas,  old  infarcts 
and  hematomas,  inspissated  pus-collections,  dermoid  cysts,  hydrocele 
fluids,  etc.;  especially  large  amounts  occur  in  the  cholesteatomatous 
tumors  of  the  ear  and  cranial  cavity. ^^ 

In  degenerative  conditions  of  the  central  nervous  system^^  choles- 
terol may  be  present  in  the  spinal  fluid  (Pighini''^),  and  in  an  old 
pleural  effusion  as  much  as  3  to 4  per  cent,  of  cholesterol  has  been  found^ 
(See  Pleural  Effusions,  Chap,  xiv.)  Windaus-  found  that  normal  aortas 
contain  about  0.15  per  cent,  cholesterol,  while  in  two  atheromatous 
aortas  he  found  1.8  per  cent,  and  1.4  per  cent.,  the  increase  being 
more  in  the  cholesterol  esters  than  in  the  free  cholesterol.  Amyloid 
kidneys,  however,  show  an  increase  only  in  the  cholesterol  esters,  and 
not  at  all  in  the  free  cholesterol.  (See  Relation  of  Lipoids  to  Fatty 
Metamorphosis,  p.  406.)  Ameseder^  found  that  28.56  per  cent,  of 
the  ether  extract  of  atheromatous  aortas  was  cholesterol.  Tlie  claim 
of  Chauffard  that  arcus  senilis,  xanthelasma,  and  other  ocular  condi- 
tions depend  on  cholesterol  deposition  is  not  substantiated  by  Mawas'* 
but  Verse^^  observed  corneal  opacity  in  rabbits  fed  cholesterol  and  oil. 
In  liquids  the  crystals  form  glistening  scales;  in  fresh  tissues  they  may 
be  recognized  by  their  solubility  in  ether,  cholorform,  hot  alcohol, 
etc.,  and  by  their  color  reactions.  In  histological  specimens  prepared 
by  the  usual  methods  the  cholesterol  is  dissolved  out,  but  the  resulting 
clear-cut  clefts  are  quite  characteristic.     In  fresh  specimens  in  which 

9*  Wells,  Jour.  Med.  Research,  1906  (14),  491. 
95  Smith  and  White,  Jour.  Path,  and  Bact.,  1907  (12),  126. 
9^  Concerning  the  chemistry  of  cholesterol  see  introductory  chapter. 
9^  See  Bostroem,  Cent.  f.  Path.,  1897  (8j,  1. 

'^  Southard  has  described  cholesterol  concretions  up  to  2  cm.  diameter  in  the 
brain  and  cord.      (Jour.  Amer.  Med.  Assoc,  1905  (45),  1731.) 
99  Riforma  Med.,  1909  (25),  67. 

1  Ruppert,  Miinch.  med.  Woch.,  1908  (55),  510;  Zunz,  Hedstrom,  and  others 
(see  Chap.  XIV). 

2  Zeit.  physiol.  Chem.,  1910  (67),  174. 
^Zeit.  physiol.  Chem.,  1911  (70),  458. 

*  Monatsbl.  f.  Augenheilk.,  1912  (13),  604. 


420  RETROGRESSIVE  CHANGES 

cholesterol  crystals  are  present,  on  treatment  with  five  parts  concen- 
trated sulphuric  acid  and  one  of  water,  the  edges  of  the  crystals  be- 
come carmine  red,  then  violet.  Concentrated  sulphuric  acid  plus  a 
trace  of  iodin  colors  the  crystals  in  sequence,  violet,  blue,  green,  and 
red.  Hirschsohn^  recommends  a  reaction  with  a  90  per  cent,  solution 
of  trichloracetic  acid  in  HCl,  which  gives  red,  then  violet,  then  blue. 
The  results  of  microchemical  examination  are  said  not  to  agree  at  all 
quantitatively  with  analytic  results.*^ 

Since  all  cells  contain  cholesterol,^  it  is  perhaps  accumulated  as  one 
of  the  least  soluble  products  of  their  disintegration.  The  origin  of 
the  normal  cell  cholesterol  is  unknown,  but  that  which  is  liberated  by 
normal  disintegration  of  cells  seems  to  be  retained  and  worked  over.^ 
It  is  not  destroyed  during  autolysis.^  Cholesterol  is  generally  con- 
sidered, but  without  convincing  proof,  to  be  a  product  of  protein  de- 
composition; if  this  is  true,  then  the  cholesterol  found  in  disintegrating 
tissues  may  be  formed  from  the  cell  proteins  during  their  decomposi- 
tion.^'' Apparently  cholesterol  crystals  may  be  slowly  removed, 
the  chief  factor  probably  being  the  giant-cells  that  are  often  found 
surrounding  them,^>  and  the  large  "foamy"  endothelial  cells  that 
take  up  especially  the  uncrystallized  cholesterol.  In  general  they 
behave  as  inert  foreign  bodies.  Xanthomatous  masses  of  various 
kinds  all  seem  to  be  composed  of  deposits  of  cholesterol  esters  which 
lead  to  proliferative  and  phagocytic  reactions  in  the  fixed  tissues.'^ 

Cholesterolemia.^' — Normal  blood  contains  0.16  to  0.17  per  cent.  (Gorham 
and  Myers)  of  cholesterol,  of  which  about  55  per  cent,  is  in  the  corpuscles,  but 
in  pathological  conditions  the  amount  in  the  plasma  varies  greatly  (Bacmeister 
and  Henes)."  Cholesterol-rich  diet  causes  a  slight  increase,'^  but  a  more  marked 
increase  is  said  to  be  obtained  in  pregnancy,"  nephritis,  early  arteriosclerosis, 
obesity,  diabetes,  and  obstructive  but  not  in  hemolytic  jaundice."  According  to 
some  observations,  in  nephritis  the  amount  of  cholesterol  bears  no  relation  to 
the  albuminuria,  and  in  uremia  it  may  be  low;  acute  febrile  diseases  usuallj'  show 
a  lowered  cholesterol,   which  is  unchanged  in  tuberculosis.     Stapp^*  describes 

6  Pharm.  Centralhalle,  1902  (43),  357. 

«  Thavscn,  Cent.  allg.  Pathol,  1015  (2()\  433. 

7  See  Doree,  Biochem.  Jour.,  1909  (4),  72. 

8  Ellis  and  Gardner,  Proc.  Royal  Soc,  London,  1912  (84),  461. 

3  Corper,  Jour.  Biol.  Chem.,  1912  (11),  37;  Shibata,  Biochem.  Zeit.,  1911  (31), 
321. 

'"  Of  historical  interest  is  Austin  Flint's  idea  that  cholesterol  in  the  blood  is 
an  important  factor  in  intoxications,  especially  in  icterus  (Amer.  Jour.  Med.  Sci., 
1862  (44),  29).     All  recent  evidence  is  to  the  effect  that  cholesterol  is  not  toxic. 

"  See  LeCount,  Jour.  Med.  Research,  1902  (7),  160;  Corper,  Jour.  Exp.  Med., 
1915  (21),  179;  Stewart,  Jour.  Path,  and  Bact.,  1915  (19),  305. 

12  Literature  given  by  Rosenbloom,  Arch.  Int.  Med.,  1913  (12),  395. 

> 3  Bibliography  by  Dewey,  Arch.  Int.  Med.,  1916  (17),  757;  Gorham  and 
Myers,  Arch.  Int.  Med.,  1917  (20),  599;  Pacini,  Med.  Record,  1919  (94),  441. 

"  Deut.  med.  Woch.,  1913  (39),  544. 

"  See  Luden,  Jour.  Biol.  Chem.,  1916  (27),  257. 

1^  The  blood  of  the  fetus  corresponds  closely  to  that  of  the  mother  in  respect 
to  free  cholesterol  but  contains  no  cholesterol  esters.  (Slemons  and  Curtis,  Amer. 
Jour.  Obst.,  1917  (75),  569.) 

"  Rothschild  and  Felson,  Arch.  Int.  Med.,  1919  (24),  520. 

"Deut.  Arch.  klin.  Med.,  191S  (127),  439;  corroborated  by  Epstein,  Amer. 
Jour.  Med.  Sci.,  1917  (154),  638. 


AMYLOID  421 

marked  cholesteroleinia  as  accompanying  severe  parenchymatous  nephritis,  but 
not  cluonic  interstitial  typos.  K'ollert  and  FinKor'""  state  that  hypercholestero- 
lemia up  to  ().2S  per  cent,  may  be  found,  but  only  when  the  kidney  is  excreting 
lipoids,  and  believe  that  the  cholesterol  is  at  least  partly  responsible  for  albuminuric 
retinitis.  Bloor,^"  however,  found  no  change  in  the  blood  cholesterol  in  nephritis. 
The  blood  content  has  been  reported  as  low  in  febrile  cutaneous  di.seases,  but 
high  in  afebrile  cutaneous  diseases  associated  with  eosinophilia.^"  However, 
DiMiis^'  states,  after  examination  of  a  largo  nundier  of  cases,  that  hypercholestero- 
loinia  was  found  only  in  diabetes,  and  that  low  cholesterol  values  are  found  in 
cachexia  or  prostration,  but  are  not  characteristic  of  anj'  particular  disease.  In 
Japan  low  values  have  been  observed  in  beriberi,  high  in  homiplegia.^'*  Numerous 
investigators  have  described  hypercholesterolemia  in  patients  with  gall-stones  (7. 
V.)  and  attribute  a  causal  relation  thereto.  The  importance  of  the  cholesterol  of 
the  blood  in  hemolysis  and  protection  therefrom  has  been  discussed  under  that 
subject;  in  anemia  there  is  usually  hypocholesterolemia. 

Experimental  hypercholesterolenua  in  animals  leads  to  a  deposition  of  choles- 
terol in  various  organs,  especially  the  aorta,*'  kidneys  and  liver,  accompanied  by 
degeneration  in  the  parenchymatous  structures,  and  excretion  of  cholesterol  in 
the  urine  and  bile;  gall-stones  may  be  formed  (Dewey).  Sometimes  lipoid-filled 
endothelial  cells  become  so  abundant  in  the  spleen  as  to  resemble  Gaucher's  dis- 
ease (Anichkov,  McMeans**).  Excessive  cholesterol  in  the  blood  reduces  phago- 
cytic activity  and  antibody  formation  in  experimental  animals.*^  Robertson  be- 
lieves cholesterol  to  have  an  accelerative  action  on  cancer  growth,  related  to  its 
hydroxyl  radical, '^^  but  in  cancer  patients  there  seems  to  be  no  cholesterolemia 
(Denis). ^^^ 

The  ratio  of  free  cholesterol  to  cholesterol  esters  in  normal  human  blood  is 
nearly  constant,  the  esters  being  about  33.5  per  cent,  in  the  blood  and  58  per  cent. 
in  the  plasma;  in  preganncy  and  during  fat  absorption  the  proportion  of  choles- 
terol esters  is  high,  in  cancer  and  nephritis  it  is  low.^^  The  blood  of  the  fetus 
contains  free  cholesterol  but  no  cholesterol  esters. 

AMYLOID28 

Virchow,  in  1853,  made  the  first  study  of  the  nature  of  the  substance 
characteristic  of  ''lardaceous"  degeneration,  and  considered  it  to  be 
a  sort  of  animal  cellulose,  because  it  often  became  blue  if  treated  with 
iodin  followed  by  sulphuric  acid.  To  this  resemblance  in  staining 
reaction  we  owe  the  unfortunate,  misleading,  but  generally  used,  name 
amyloid."^     It  was  but  a  few  years   (1859)   before  Friedreich  and 

»»"  Mtinch.  med.  Woch.,  1918  (65),  816. 

1^  Jour.  Biol.  Chem.,  1917  (31),  575;  also  Kahn,  Arch.  Int.  Med.,  1920  (25),  112. 

2"  Fischl,  Wien.  klin.  Woch.,  1914  (27),  982. 

"  Jour.  Biol.  Chem.,  1917  (29),  93. 

22  Bull.  Naval  Med.  Assoc,  Japan,  Feb.,  1919. 

"  See  Adler,  Trans.  Assoc.  Amer.  Phys.,  1917  (32),  255. 

24  Jour.  Med.  Res.,  1916  (33),  481.  The  material  in  the  cells  in  Gaucher's 
disease  is  perhaps  a  protein-phosphatid  compound  (Mandelbaum  and  Downey, 
Fol.  Hematol.,  1916  (20),  139. 

2^  Dewey  and  Nuzum,  Jour.  Infect.  Dis.,  1914  (15),  472. 

"  Jour.  "Cancer  Res.,  1918  (3),  75. 

^^^  Luden  reports  an  increase  (Jour.  Lab.  Clin.  Med.,  1916  (1),  662. 

"  Bloor  and  Knudson,  Jour.  Biol.  Chem.,  1917  (29),  7;  1917  (32),  337. 

-8  General  literature  to  1893,  see  Wichmann,  Ziegler's  Beitr.,  1893  (13),  487; 
also  Lubarseh,  Ergeb.  allg.  Path.,  1897  (4),  449;  discussion  in  the  Verh.  Deut. 
Path.  Gesellsch.,  1904  (7),  2-51;  Davidsohn,  Virchow's  Arch.,  1908  (192),  226, 
and  Ergebnisse  allg.  Path.,  1908  (12),  424. 

-^  In  view  of  the  fact  that  this  substance  is  chemically  related  to  chondrin, 
and  that  it  also  closely  resembles  this  substance  physically,  it  has  seemed  to  the 
writer  that  the  name  '"'chondroid"  would  be  much  more  appropriate  than  any  of 
the  many  more  or  less  misleading  and  inappropriate  titles  that  are  at  present  in 
use.  The  very  multiplicity  of  these  terms,  however,  prohibits  any  attempt  to 
introduce  still  another.  A  particularly  unfortunate  source  of  confusion  exists 
in  the  use  of  the  name  amyloid  for  a  vegetable  substance,  formed  by  the  action 
of  acids  upon  cellulose. 


422  RETROGRESSIVE  CHANGES 

Kekule  showed  that  the  substance  in  question  was  of  protein  nature; 
their  methods  were  very  crude,  but  the  main  fact  was  soon  better 
substantiated  by  Klihne  and  Rudneff  (1865).  Krawkow,^''  however, 
in  1897  gave  us  the  first  good  idea  of  the  composition  of  amyloid  sub- 
stance through  his  ampKfication  of  Oddi's^^  observation  that  amyloid 
organs  contain  chondroitin-sulphuric  acid,  finding  that  amyloid  is  a 
compound  of  protein  with  this  acid,  similar  to  nucleoprotein,  which 
is  a  compound  of  nucleic  acid  and  protein.  This  work  has  received 
general  acceptance,  although  a  later  paper  by  Hanssen^^  reports  a 
study  of  amyloid  material  isolated  in  pure  condition  from  sago  spleens 
by  mechanical  means,  which  contained  no  chondroitin-sulphuric  acid, 
although  the  amyloid  organs  taken  in  toto  do  contain  ah  excess  of  sul- 
phur as  sulphate.  This  important  contradiction  to  prevailing  ideas 
has  not,  so  far  as  I  can  find,  been  subjected  to  investigation  by  others, 
with  the  exception  of  a  casual  remark  by  Mayeda^^  that  a  prepara- 
tion of  amyloid  which  he  had  made  did  not  yield  sulphuric  acid. 

Chondroitin-sulphuric  acid,  which  has  been  studied  especially  by  Morner  and 
by  Schmiedeberg,^^  has  the  formula  C18H27NSO17,  according  to  tlie  latter,  and 
yields  on  cleavage  chondroitin  and  sulphuric  acid,  as  follows. 

Ci8H27NSOn+H20  =  C18H27NO14+H2SO4 
Kondo,^^  however,  gives  it  an  empirical  formula  of  C15H27NSO16,  there  being  ap- 
parently two  equivalents  of  the  base  for  each  SO4  group.  Levene  and  La  Forge'^ 
have  demonstrated  that  chondroitin-sulphuric  acid  consists  of  sulphuric  acid, 
acetic  acid,  chondrosamine  which  is  an  isomer  of  glucosamine,  and  glucuronic 
acid.  It  unites  with  histones  and  forms  a  precipitate.^^  Chondroitin  is  a  gummy 
substance  which  in  turn  may  be  split  into  acetic  acid  and  a  reducing  substance, 
chondrosin.  Chondroitin-sulphuric  acid  is  the  characteristic  component  of  car- 
tilage, but  it  is  also  found  in  the  walls  of  the  aorta  and  other  elastic  structures 
(Krawkow).  It  has  also  been  found  in  a  uterine  fibroma  and  in  bone  tissue  by 
Krawkow,  but  could  not  be  found  in  the  parenchymatous  organs,  normal  and 
pathological,  or  in  chitinous  structures.     Morner  has  also  found  it  in  a  chondroma. 

Chemistry  of  Amyloid. — Krawkow  separated  amyloid  from  nu- 
cleoprotein, to  which  it  is  most  closely  related,  by  dissolving  both  sub- 
stances from  the  minced  amyloid  organs  with  ammonia,  precipitating 
with  acid,  and  then  taking  up  the  amyloid  with  Ba  (OH)  2  solution,  in 
which  the  nucleoprotein  does  not  dissolve.  Amyloid  thus  isolated  is 
a  nearly  white  powder,  which  is  easily  soluble  in  alkalies,  but  slightly 
in_^acids,  and  is  very  resistant  to  pepsin  digestion.  The  elementary 
composition  was  found  by  Krawkow  to  be  approximately  as  follows : 
C  =  49-50%;  H  =  6.65-7%;  N  =  13.8-14%;  S  =  2.65-2.9%;  P  in 
traces  only. 

^»  Arch.  exp.  Path.  u.  Pharm.,  1897  (40),  196. 

31  Ibid.,  1894  (33),  377. 

32  Hiochem.  Zcit.,  1908  (13),  185. 

"  Zeit.  physiol.  Chem.,  1909  (58),  475. 

3*  Morner,"  Skand.  Arch.  Physiol.,  1889  (1),  210;  Zcit.  physiol.  Chem.,  1895 
(20),  357,  and  1897  (23),  311;  Sciimiodeberg,  Arch.  exp.  Path.  u.  Pharm.,  1891 
(28),  358.  Hoc  also  Levene  and  La  Forge,  Jour.  Biol.  Chom.,  1913  (15),  09  and 
155;  1914  (18),  123. 

•••f'  Biocliem.  Zeit.,  1910  (2G),  116. 

3«  Pons,  Arch,  internat.  physiol.,  1909  (8),  393. 


AMYLOID 


423 


Quite  similar  analytic  results  have  been  obtained  by  Neuberg,'^ 
who  corroborated  Krawkow's  finding  of  a  body  of  apparently  similar 
composition  in  tlie  normal  aorta.  Neuberg  has  studied  especially  the 
protein  constituent  of  the  amyloid  compound,  and  found  it  character- 
ized by  a  high  proportion  of  diamino-nitrogen,"^  as  compared  with 
most  proteins,  as  shown  in  the  following  table  giving  the  percentage 
of  the  total  N  contained  in  each  of  the  three  forms,  amid-nitrogen 
(ammonia),  monamino-acids,  and  diamino-acids: 


Tahle  I 


Monamino- 

acid 

nitrogen 


Diamino- 

acid 
nitrogen 


Amid 
nitrogen 


Liver  amyloid .  .  .  . 
Spleen  amyloid .  .  . 
Aorta  "amyloid". 

Gelatin 

Casein 


43.2 
30.6 
54.9 
62.5 
76.0 


51.2 
57.0 
36.0 
35.8 
11.1 


4.9 

11.2 

8.8 

1.6 

13.4 


The  variations  in  the  composition  of  the  different  amyloids,  as 
shown  in  the  above  table,  indicate  that  the  protein  group  may  vary  in 
different  organs  in  different  cases,  and  also  indicate  that  the  "amyloid- 
like" substance  of  normal  vessels  is  not  the  same  as  the  pathological 
substance.  Corresponding  variations  were  found  in  the  apportion- 
ment of  the  sulphur  between  that  which  is  in  the  form  of  oxidized  sul- 
phur and  the  unoxidized  sulphur.  The  proportion  of  the  different 
amino-acids  in  the  protein  constituent  of  amyloid  is  strikingl}'  like  that 
of  thjmius  histon,  and  entirely  dissimilar  to  the  apparently  closely 
related  elastin,  as  shown  by  Table  II. 

Table  II 


Cleavage  products  (in  percentages) 


Amyloid 


Elastin 


Thymus 
histon 


Glycocoll 0.8  !        25.8 

Leucine 22.2  j        45.0 

Glutaminic  acid 3.8                  0.7 

Tyrosine 4.0  i          0.3 

a-Proline 3.1                  1.7 

Arginine - 13.9                  0.3 

Lj'sine 11.6  I         .... 


0.5 

11.8 
3.7 
5.2 
1.5 

14.5 
7.7 


"Verh.  Deut.  Path.  GeselL,  1904  (7), 19. 

^^  Corroborated  by  Jackson  and  Pearce  (Jour.  Exp.  Med.,  1907  (9),  520),  but 
not  by  Mayeda  (Zeit.  physiol.,  Cliem.,  1909  (58),  469),  who  found  histidine,  which 
Neuberg  had  missed,  and  a  lower  arginine  and  lysine  content  than  histon  re- 
quires. 


424  RETROGRESSIVE  CHANGES 

This  carries  out  the  resemblance  of  amyloid  to  nucleoproteins,  and, 
likewise,  Neuberg  found  amj-loid  very  slowly  digested  bj^  pepsin,  and 
much  better  by  trypsin,  although  less  rapidly  than  simple  protein;  it 
is  also  destroyed  by  autolytic  enzymes,  for  amyloid  tissues  readily 
undergo  autolysis. ^^  Neuberg  considers,  from  the  above  results,  that 
amyloid  is  probably  a  transformation-product  of  the  tissue  protein, 
similar  to  the  transformation  of  simple  proteins  into  protamins  that 
occurs  in  the  testicle  of  spawning  salmon  as  they  go  up  the  streams, 
as  shown  by  Miescher's  classical  studies.  Raubitschek'*°  found  that 
isolated  amyloid,  when  used  for  immune  reactions,  behaved  like  a 
specific  protein,  different  from  the  normal  proteins  of  the  animal  from 
whence  it  came  and  apparently  biologically  the  same  in  different  spe- 
cies.    (This  observation  awaits  confirmation.) 

Krawkow  considers  that  amyloid  differs  from  normal  chondroitin- 
sulphuric  acid  compounds,  such  as  cartilage,  in  that  in  the  latter  the 
acid  radical  is  in  a  loose  combination  with  the  protein,  while  in  amy- 
loid the  combination  is  a  very  firm  one,  perhaps  in  the  nature  of  an 
ester.  The  occurrence  of  the  typical  amyloid  reaction  in  what  ap- 
pears otherwise  to  be  normal  cartilage,  occasionally  observed  in  senile 
tissues,  may  be  due  to  the  transformation  of  loosely  bound  into  firmly 
bound  chondroitin-sulphuric  acid.  In  any  event,  amyloid  is  not  essen- 
tially a  pathological  product,  but  rather  a  slightly  modified  normal 
constituent  of  the  body.  However,  in  view  of  the  contradictory  results 
of  Hanssen  and  Mayeda,  as  yet  uncontroverted,  the  chemical  nature  of 
amyloid  must  be  considered  as  undetermined.  An  important  con- 
sideration is  that  amyloid  deposition  occurs  under  similar  conditions 
in  all  sorts  of  animals,  including  birds;  it  is  very  often  found  in  the 
livers  of  antitoxin  horses,  and  mice  are  especially  prone  to  a  severe 
amyloidosis  after  relatively  slight  and  brief  infectious  processes. ^^ 

Staining  Properties. — The  classical  reaction  for  amyloid  is  its  staining  a  reddish 
brown  when  treated  with  iodin  (best  as  Lugol's  solution)  in  the  fresh  state.  Such 
stained  specimens,  if  afterward  treated  with  dilute  sulphuric  acid,  usually  become 
blue  or  greenish,  but  may  merely  turn  a  deeper  brown.  Occasionally  old  compact 
amyloid  may  stain  bluish  or  green  with  iodin  alone.  The  iodin  reaction  disappears 
in  specimens  that  have  been  kept  for  some  time  in  preserving  fluids,  or  in  tissues 
that  have  become  alkaline,  and  is  generally  less  persistent  than  the  metachromatic 
staining  by  methyl-violet  or  methyl-green,  which  color  the  amyloid  red.  Oc- 
casionally an  otherwise  typical  amyloid  will  fail  to  react  to  iodin,  but  will  stain 
well  witii  methyl-violet.  All  these  variations  may  occur  in  different  specimens 
from  the  same  body,  and  the  blue  iodin-sulphuric  acid  reaction  is  usually  given 
well  only  bj'  splenic  amyloid.  These  variations  probably  dejjcnd  upon  the  age 
and  stage  of  development  of  the  amyloid,  or  upon  secondary  alterations,  and  are 
perhaps  related  to  Neuberg's  observations  on  the  difference  in  -composition  of 
amyloid  of  different  origins. 

Krawkow  studied  these  reactions  with  pure,  isolated  amyloid,  and  found 
evidence  that  the  iodin  reaction  depends  upon  the  physical  properties  of  the 

^*  Concerning  the  absorption  of  amyloid  see  Dantchokow,  Virchow's  Archiv., 
1907  (187),  1. 

"Verh.  Dcut.  Path.  Gesell.,  1910  (14),  273. 

"  See  Finzi,  Lo  Speriment.,  1911  (05),  483;  Davidsohn,  Virchow's  Arch.,  1908 
(192),  22(). 


AMYLOID  425 

amyloid,  while  the  inethyl-violct  stain  is  a  chemical  reaction,  and  hence  the  iodin 
reaction  is  much  the  more  readily  altered  or  lost.  As  Dickinson^- says,  amyloid 
stains  with  iodin  simph'  as  if  it  absorbed  the  iodin  more  than  does  the  surrounding 
tissue.  Krawkow  believed  that  the  methyl-violet  reaction  is  due  to  the  dye  forming 
a  compound  with  the  chondroit in-sulphuric  acid,  for  he  found  that  these  substances 
unite  with  one  another  to  form  a  rose-red  precipitate.  Hanssen,  however,  holds 
that  the  dj'es  react  with  the  protein,  the  iodin  with  some  other,  unknown  labile 
substance.  Schmidt  found  that  implanted  pieces  of  amyloid  lost  their  iodin  reac- 
tion as  they  underwent  digestion,  while  the  methyl-violet  reaction  was  still  very 
distinct. ■•*  It  is  evident,  therefore,  that  iodin  is  not  by  itself  a  specific  stain  for 
amyloid,  especially  as  glycogen  gives  a  similar  reaction,**  while  true  amyloid  may 
not   react. 

Leupold**  summarizes  his  investigations  as  follows:  Amyloid  is  a  complex 
of  different  substances  which  are  differentiated  by  micro-chemical  reactions. 
The  protein  ground  substance  of  the  amyloid  is  refractory  to  the  typical 
amyloid  reactions.  The  group  which  is  responsible  for  the  methyl-violet  reaction 
is  intimately  combined  with  this  protein  substance  and  is  separated  from  it  only  by 
the  action  of  alkali.  The  groups  which  give  respectively  the  iodin  and  the  iodin- 
sulphuric  acid  reactions  are  closely  related  to  each  other.  Nevertheless  the  iodin- 
sulphuric  acid  reaction  is  a  completely  independent  one  and  is  not  a  modification 
of  the  iodin  reaction.  The  occurrence  of  different  colors  in  the  iodin-sulphuric 
acid  reaction  depends  upon  different  degrees  of  oxidation.  Amyloid  is  an  emulsion 
colloid  in  the  gel  state.  After  oxidation  with  potassium  permanganate  it  is 
soluble  in  ammonia,  NaOH  and  Ba  (OH) 2.  Conjugated  sulphuric  acid  plays  an 
important  part  in  the  production  of  amyloid  in  the  organism.  The.existence 
of  large  amounts  of  conjugated  sulphuric  acid  produces  amyloid  which  gives  the 
iodin  reaction.  The  methjd-violet  reaction  also  depends  on  the  presence  of  con- 
jugated sulphuric  acid;  however,  for  its  production  there  must  probably  occur  a 
reduction  in  the  amyloid  protein.  The  group  which  gives  the  iodin-sulphuric  acid 
reaction  occurs  through  decomposition  and  perhaps  does  not  depend  upon  the 
sulphuric  acid. 

The  Origin  of  Amyloid 

This  question  has  not  been  at  all  cleared  up  as  yet  by  the  advances 
made  in  our  knowledge  of  the  chemistry  of  amyloid  substance.  The 
fact  that  chondroitin-sulphuric  acid  is  a  characteristic  constituent 
suggests  that  this  bodj^  niay  be  liberated  in  considerable  amount  dur- 
ing the  destructive  processes  to  which  amyloidosis  is  usually  sec- 
ondary; this  idea  is  further  supported  by  the  fact  that  amyloidosis 
occurs  particularly  after  chronic  suppuration  in  bone  and  lungs,  both 
of  which  tissues,  according  to  Krawkow,  contain  chondroitin-sulphuric 
acid.  This  idea  was  not  substantiated,  however,  by  the  experiments 
made  by  Oddi  and  by  Kettner,*^  who  fed  and  injected  into  animals 
large  quantities  of  the  sodium  salt  of  chondroitin-sulphuric  acid  with- 
out producing  amyloid  changes.  Unpublished  experiments  of  the 
writer  with  the  same  material,  as  well  as  with  ground-up  cartilage  and 
with  mucin,  were  equally  unsuccessful.    Likewise  mice  injected  by 

"  Allbutt's  System,  vol.  3,  p.  225. 

"Litten  (Verh.  Deut.  Path.  GeselL,  1904  (7),  47)  states  that  thionin  and 
kresyl-violet  are  the  most  specific  stains  for  amyloid,  which  they  color  blue; 
whereas  methyl-violet  stains  red  not  onlv  amyloid  but  also  mucin,  mast  cell 
granules,  and  the  ground  substance  of  cartilage.  V.  Gieson's  stain  usually  colors 
amyloid  pale  yellow,  and  hyalin  red. 

**  See  Wichmann,  Ziegler's  Beitr.,  1893  (13),  487. 

"  Beitr.  path.  Anat.,  1918  (64),  347. 

«  Arch.  exp.  Path.  u.  Pharm.,  1902  (47),  178. 


426  RETROGRESSIVE  CHANGES 

Strada^^  with  the  nucleoprotein  of  pus,  the  so-called  pyin,  or  with 
chrondroitin-sulphuric  acid,  did  not  develop  amyloidosis.  Oestreich^* 
injected  cancer  patients  with  chondroitin-sulphuric  acid  for  thera- 
peutic purposes,  but  no  amyloidosis  resulted.  As  it  is  possible  to 
cause  amyloidosis  experimentally  in  animals,  especially  chickens  and 
rabbits,  by  causing  protracted  suppuration  or  chronic  intoxication 
with  bacterial  filtrates,  these  negative  results  speak  strongly  against 
the  idea  of  a  transportation  of  chondroitin-sulphuric  acid,  but  do  not 
determine  it  finally.  They  may  also,  with  propriety,  be  used  in  sup- 
port of  the  statement  of  Hanssen  that  amyloid  does  not  contain  chon- 
droitin-sulphuric acid.  Leupold"*^  advances  the  following  hypothesis: 
In  chronic  suppuration  a  soluble  protein  circulates  in  the  blood  which 
stimulates  the  formation  of  "defensive  ferments."  This  protein 
substance,  under  certain  conditions,  is  deposited  in  organs  where 
large  amounts  of  sulphuric  acid  occur.  For  the  development  of 
amyloid  there  are  necessary  three  factors:  A  preformed  protein,  an 
increased  amount  of  conjugated  sulphuric  acid,  and  an  inefficiency  of 
the  amyloid-filled  organ  to  eliminate  the  increased  amount  of  conju- 
gated sulphuric  acid. 

There  is  usually  much  difficulty  in  producing  amyloid  experiment- 
ally, for  in  only  a  certain  proportion  of  cases  are  the  experiments  posi- 
tive (in  but  about  one-third  of  Davidsohn's'*'*  100  trials,  and  many 
other  experimenters  have  been  much  less  successful). ^°  Davidsohn, 
faihng  always  to  get  amyloid  experimentally  after  the  spleen  had  been 
removed,  suggests  that  this  organ  (in  which  amyloid  is  usually  earliest 
and  most  abundantly  observed)  produces  an  enzyme,  which  causes  a 
precipitation  of  amyloid  in  the  tissues  from  a  soluble  precursor  brought 
in  the  blood  from  the  site  of  cell  destruction.  Schmidt^^  considers  it 
probable  that  some  enzymatic  action  causes  a  precipitation  or  coagula- 
tion of  the  substance  in  the  tissue-spaces  or  lymph-vessels.  Amyloid 
is  never  deposited  in  the  cells  themselves, ^^  and  it  seems  to  be  now 
generally  considered  that  the  amyloid  material  is  infiltrated  in  the 
form  of  a  soluble  modification  or  precursor  and  that  it  is  not  manu- 
factured in  the  organ  where  it  is  found.  It  is  an  interesting  fact  that 
a  practically  identical  substance  is  formed  in  all  tissues  and  in  al 
species  of  animals,  even  when  the  cause  is  quite  difi"erent.  Whether 
the  precursors  are  brought  to  the  organ  in  solution,  or  in  leucocytes,  is 
unknown — probably  the  former.  Pollitzer^^  states  that  in  various 
infections,  especially  coccus  infections,  chondroitinsulphuric  acid  is 
excreted  in  the  urine;  if  this  is  correct  it  has  an  undoubted  bearing  on 

*'  Hiochein.  Zeit.,  1909  (16j,  195. 
"Zeit.  Krebsforsch.,  1911  (11),  44. 
"  Verb.  Dent.  Path.  Gesell.,  1904  (7),  39. 

»"  See  Tarclietti,  Deut.  Arch.  klin.  Mod.,  1903  (75),  526.     Hirosc,  Bull.  Johns 
Hop.  Hosp.,  1918  (29),  40. 

*'  Verh.  Deut.  Path.  Gesoll.,  1904  (7),  2. 

"  See  Kbert,  Virchow's  Arch.,  1914  (210),  77. 

"  Deut.  mod.  Woeh.,  1912  (3S),  1538. 


HYALINE  DEGENERATION  427 

the  genesis  of  amyloidosis.  The  presence  of  glyeothionic  acid  in  pus'* 
is  of  similar  significance.  The  hypothesis  that  amyloid  is  formed  from 
disintegrating  red  corpuscles  is  probal)!}^  incorrect.  Amyloidosis  is 
produced  by  the  most  varied  species  of  bacteria  and  by  their  toxins, 
although  the  staphylococcus  is  usually  most  effective  in  experimental 
work.^^  Neither  is  suppuration  absolutely  essential,  for  injection  of 
toxins  alone  (c.  g.,  in  preparing  diplit  heria  antitoxin'^*"'),  without  suppura- 
tion, may  produce  amyloidosis,  as  also  frequently  does  syphilis  without 
suppuration  and,  less  often,  many  other  non-suppurative  conditions 
(e.  g.,  tumors).  Wago^^  reports  finding  a  widespread  "amyloid-like" 
degeneration  in  rabbits  immunized  with  sterile  pancreatic   extracts. 

Local  amyloid  accumulations  are  of  some  interest  in  considering  the  genesis  of 

the  usual  generalized  form.  They  occur  particularly  as  small  tumors  in  the  larynx, 
bronchi,  nasal  septum,  and  eyelids;  as  all  these  tissues  are  normally  rich  in  chon- 
droitin-sulphuric  acid,  it  seems  probable  that  the  amjdoid  arises  from  a  local 
overproduction  of  chondroitin-sulphuric  acid,  which  becomes  bound  with  proteins 
in  si(u.  This  makes  it  seem  more  probable  that,  in  spite  of  the  lack  of  positive 
experimental  evidence,  general  amyloidosis  is  due  to  liberation  of  excessive  quanti- 
ties of  chondroitin-sulphuric  acid  in  the  sites  of  tissue  destruction. 

Another  form  of  local  amyloid  is  seen  particularly  in  the  regional  lymph-glands 
of  suppurating  areas;  e.  g.,  the  lumbar  glands  in  vertebral  caries,  the  axillary  glands 
in  shoulder-joint  suppuration.  This  local  amyloidosis  is  undoubtedly  due  simply 
to  the  fact  that  these  glands  receive  first,  and  in  largest  amounts,  the  cau.se,  what- 
ever it  may  be,  of  the  amyloid  production.*^  Less  readily  explained  are  cases  of 
extensive  amyloidosis  limited  to  the  heart.*' 

Corpora  amylacea  will  be  found  discussed  under  "Concretions"  (Chap.  xvii). 

HYALINE  DEGENERATION*" 

Much  confusion  concerning  this  condition  may  be  avoided  if  we  ap- 
preciate that  the  term  hyaline  indicates  a  certain  physical  condition, 
which  may  be  exhibited  by  many  substances  of  widely  different  na- 
ture and  origin.  There  is  7io  one  chemical  compound,  ^'hyalin," 
which,  accumulating  in  cells  or  tissues,  produces  a  hyaline  appear- 
ance. The  limits  of  the  application  of  the  term  "hyaline  degenera- 
tion," even  to  histological  findings,  is  not  agreed  upon,  but  in  general 
it  is  used  to  apply  to  clear,  homogeneous,  pathological  substances 
that  possess  a  decided  affinity  for  acid  stains,  such  as  eosin.  Some- 
what similar  substances,  usually  of  epithelial  origin,  which  do  not 

"  Mandel  and  Levene,  Biochem.  Zeit.,  1907  (4),  78. 

**  In  a  series  of  experiments  directed  to  ascertain,  if  possible,  which  constituent 
of  pus  might  be  the  cause  of  amyloid  formation,  1  was  unable  to  secure  amyloid 
by  protracted  intoxication  of  rabbits  by  Witte's  "peptone,"  which  consists  chiefly 
of  proteoses  (Trans.  Chicago  Path.  Soc,  1903  (5),  240). 

**  See  Lewis,  Jour.  Med.  Research,  1906  (15),  -449. 

"Arch.  Int.  Med.,  1919  (23),  251. 

*^  Quite  unexplained  is  the  cause  of  the  rarely  observed  localization  of  amyloid 
in  the  wall  of  the  urinary  bladder.  See  Lucksch  (Verb.  Deut.  path.  Gesell.,  1904 
(7),  34).  ConcretioiLs  giving  the  amvloid  reactions  have  been  found  in  the  pelvis 
of  the  kidney.  (Schmidt,  Cent.  f.  Pathol.,  1912  (23),  865.  Mivauchi,  ibid.,  1915 
(26),  289.) 

*9  See  Hecht.  Virchow's  Arch.,  1910  (202),  168. 

*"  General  literature,  seeLubarsch,  Ergeb.  allg.  Path.,  1897  (4),  449. 


428  RETROGRESSIVE  CHANGES 

take  either  acid  or  basic  stains  strongly,  are  usually  called  "colloid." 
We  may  properly  consider  that  pathological  hyalin  can  be  divided 
into  two  chief  classes  according  to  its  origin:  (1)  connective-tissue 
hyahn;  (2)  epithelial  hyahn. 

Connective=tissue  hyalin  is  characterized,  like  amyloid,  by  be- 
ing deposited  in  or  among  the  fibrillar  substance  of  connective  tissues, 
and  not  within  the  cells  themselves,  but  there  are  undoubtedly  several 
different  sorts  of  chemical  substances  responsible  for  various  forms  of 
connective-tissue  hyalin.  One  form  is  closely  associated  with  amyloid, 
being  found  in  organs  showing  amyloid  degeneration,  or  in  other  tis- 
sues in  the  same  body.  In  experimentally  produced  amyloidosis  in 
animals  it  has  been  shown  that  such  a  hyahne  substance  may  appear 
before  the  amyloid,  which  eventually  replaces  it;  hence,  it  has  been 
suggested  that  hyalin  is  a  precursor  of  amyloid. ^^  Such  hyalin  differs 
from  true  amyloid  only  in  its  failure  to  give  the  characteristic  stain- 
ing reaction  of  amyloid;  in  all  other  respects,  e.  g.,  cause,  location, 
termination,  it  is  the  same.  As  it  has  been  shown  (see  preceding  sec- 
tion) that  the  staining  properties  of  amyloid  are  very  inconstant,  it 
is  probable  that  the  above-described  variety  of  hyahn  is  merely  an  in- 
completely developed,  or  occasionally  a  retrogressively  altered  amy- 
loid. However,  it  is  probably  not  necessary,  as  some  authors  have 
thought,  that  amyloid  should  always  pass  through  this  hyaline  stage 
in  its  formation. 

Quite  different,  without  doubt,  is  the  form  of  hyalin  observed  in 
scar  tissue.  This  variety  develops  almost  constantly  in  any  scar-tissue 
after  the  blood-supply  has  been  reduced  to  a  minimum  through  con- 
traction, and  is  seen  characteristically  in  the  corpora  fibrosa  of  the 
ovary,  fibroid  glomerules  in  chronic  nephritis,  thickened  pleural,  peri- 
cardial, and  episplenitis  scars,  etc.  Such  hyahne  substance  occurs 
independent  of  the  usual  causes  of  amyloid,  affects  only  abnormal 
fibrous  tissue,  never  changes  into  amyloid,  and  is  prone  to  undergo 
calcification — it  surely  has  no  close  chemical  relation  to  the  form  of 
hyalin  that  does  become  amyloid.  Presumably,  it  is  similar  in  na- 
ture to  the  collagen  of  normal  fibrous  tissue  intercellular  substance, 
which  has  undergone  physical  rather  than  chemical  changes  into  a 
homogeneous  hyaline  substance.  For  its  physiological  prototype  it 
has  the  thick  "collagenous"  fibers  of  the  subcutaneous  connective  tis- 
sue. 

Probably  of  quite  different  origin  is  the  hyalin  that  develops  from 
elastic  tissue,  as  seen  best  in  the  thick-walled,  partly  obliterated 
arteries  of  the  senile  spleen;  and  less  characteristically  in  the  early 
stages  of  arteriosclerosis,  since  here  the  preceding  form  of  connective- 
tissue  hyalin  may  also  occur.  Although  arterial  elastic  tissue  is 
related  chemically  to  amyloid,  these  hj^alinc  vessels  do  not  develop  the 
usual  amyloid  reaction,  but  retain  more  or  less  of  the  specific,  clastic 

"  SeeLubarsch,  Cent.  f.  Pathol.,  1910  (21),  97. 


COLLOID  DFXiENERATION  429 

tissue  stains.  Presumably  (his  I'oiiii  ol"  hyalin  is  an  increased  and 
physically  altered  elastin.**- 

Epithelial  hyalin  occurs  within  the  cells,  and  includes  substances 
of  presumably  widely  diverse  chemical  nature,  from  the  keratin  of 
squamous  epithelium  to  the  small  intracellular  hyahne  granules  of 
carcinoma  and  other  degenerating  cells  (Russell's  fuchsin  bodies)/'^ 
Fuchsin  bodies  are  found  also  in  plasma  cells  and,  less  often,  in  other 
cells,  including  granulation  tissue;  the  fuchsin  bodies  of  this  class  are 
beheved  by  Brown^^  to  be  derived  from  red  corpuscles,  a  view  also 
held  by  Saltykow,  but  not  accepted  by  all  pathologists/'^  Extracellu- 
lar substances  of  hyaline  character,  but  of  unknown  composition,  may 
also  be  produced  by  epithelium,  e.  g.,  hyaline  casts  in  the  renal 
tubules. 

The  composition  of  none  of  these  forms  of  hyalin  is  known,  except 
that  by  using  microchemical  methods  Unna®®  has  found  evidence  that 
keratohyalin  consists  of  two  elements,  one  of  acid  character,  appar- 
enth'  derived  from  the  chromatin,  and  a  basic  substance  resembling 
the  globulins. 

Many  other  pathological  materials  of  widely  differing  nature  may, 
under  certain  conditions,  assume  a  hyaline  appearance;  e.  g.,  fibrinous 
exudates  and  thrombi,  degenerated  muscle-fibers  (Zenker's  or  "waxy" 
degeneration),  tumor-cells,  (cylindroma),  etc.  In  all  of  these  the 
chemical  nature  of  the  parent  substance  or  substances  is  probably 
much  less  altered  than  its  physical  appearance,  but  whether  the  change 
is  related  to  the  process  of  protein  coagulation  or  not  is  unknown. 
Occasionally  hyalin,  both  in  epithelium  and  connective-tissue,  takes 
on  a  crystalline  structure  (Freifeld).^^ 

COLLOID  DEGENERATION 

This  term,  also,  has  a  very  indefinite  meaning,  and  is  applied  to 
many  different  conditions  by  various  authors.  Thus,  v.  Reckhng- 
hausen  includes  under  this  name  amyloid,  epithelial  hyaline,  and  mu- 
coid degeneration.  Marchand  includes  hyaline  connective-tissue 
degeneration,  and,  also,  as  do  most  other  writers,  the  mucoid  degenera- 
tion of  carcinoma.  Ziegler  rightly  protests  against  the  inclusion  of 
mucin  under  this  heading,  but  includes  the  corpora  amylacea.  On  ac- 
count of  the  chscovery  by  Baumann  of  the  specific  chemical  nature  of 
thyroid  colloid  it  becomes  particularly  unfortunate  that  the  term 
''colloid"  has  such  a  wide  and  uncertain  apphcation.  It  would  seem 
that  the  safest  view  to  take  is  that  the  word  colloid  is  merely  morpho- 

"  See  Schmidt,  Verb.  Deut.  path.  Gesell.,  1904  (7),  2. 

*^  Literature,  see  Hektoen,  Progressive  Med.,  1899  (ii),  241. 

6*  Jour.  Exp.  Med.,  1910  (12),  533. 

«5  See  discussion,  Verh.  Deut.  path.  Gesell.,  1908  (12),  265;  Miinter,  Virchow's 
Arch.,  1909  (198),  105. 

«  Berl.  klin.  Woch.,  1914  (51),  598. 

"Ziegler's  Beitr.,  1912  (55),  168;  also  Goodpasture,  Jour.  Med.  Res.,  1917 
(35),  259. 


430  RETROGRESSIVE  CHANGES 

logically  and  macroscopically  descriptive  of  certain  products  of  cell 
activity  or  disintegration,  which  have  nothing  in  common  except  the 
fact  that  they  form  a  thick,  glue-like  or  gelatinous,  often  yellowish  or 
brownish  substance.  There  is  7io  one  definite  substance  colloid,  accord- 
ing to  the  usual  usage  of  the  word  in  pathological  literature,  but  many 
different  protein  substances  may  assume  the  appearance  to  which  the 
name  "colloid"  is  given.  Looking  at  the  matter  in  this  way,  we  must 
recognize  as  the  usual  "colloid"  substances,  the  following  chemical 
bodies: 

Thyroid  colloid,  the  physiological  prototype  of  the  group.  This  consi.sts  of 
a  compound  of  globulin  with  an  iodin-containing  substance,  thj^roiodin,  the  com- 
pound protein  being  called  by  Oswald  iodothyreoglobulin.  It  occurs  pathologi- 
cally only  in  cystic  and  similar  changes  in  the  thyroid  or  accessory  thyroids.  Being 
a  specific  product  of  the  thyroid  with  definite  physiological  properties,  it  manifestly 
has  only  a  morphological  relation  to  the  other  forms  of  colloid  found  in  degenerating 
tumors,  etc.  In  cysts  of  the  thyroid,  and  less  often  in  tumors,  there  is  occasionally 
found  a  more  dense  "colloid"  material  of  deeper  color,  the  "caoutchouc  colloid" 
of  the  Germans;  this  seems  to  result  largely  from  transformation  of  red  corpuscles 
in  hemorrhagic  cysts  (Wiget).**  (The  nature  of  thyroid  colloid  is  discussed  more 
fully  under  "  Diseases  of  the  Thyroid,"  Chap,  xxii.) 

Mucin,  when  secreted  in  closed  cavities,  as  in  tumors,  where  it  becomes  thick- 
ened by  partial  absorption  of  the  water,  may  take  on  a  "colloid"  appearance  while 
retaining  its  chemical  and  tinctorial  characteristics.  This  is  particularly  observed 
in  the  "colloid"  carcinomas  which  arise  especially  from  the  mucous  membrane  of 
the  alimentary  tract.  This  substance  is,  of  course,  quite  specific  both  in  its  chem- 
ical nature  and  its  origin  from  specialized  epithelial  cells,  and  the  process  should 
properly  be  considered  as  a  "mucoid  degeneration." 

Pseudomucin,  which  differs  from  mucin  in  not  being  precipitated  by  acetic  acid, 
is  a  common  component  of  ovarian  c.ysts,  and  when  somewhat  concentrated  by 
absorption  of  water,  forms  "typical  colloid."  Because  it  is  alkaline,  this  form  of 
colloid  tends  to  stain  rather  with  the  acid  dyes  (eosin,  acid  fuchsin,  etc.),  while 
true  mucin  stains  with  basic  dyes.  Several  varieties  of  pseudomucin  have  been 
described  by  Pfannenstiel,  and  their  properties  will  be  considered  more  fully  in 
the  section  on  "Ovarian  Tumors"  (Chap.  xix).  The  clear,  glassy,  yellowish  sub- 
stance contained  in  small  cavities  of  ovarian  tumors,  which  is  usually  called 
"colloid,"  consists  of  nearly  pure  pseudo-mucin.  All  these  substances  yield  a 
reducing  substance  on  boiling  with  acids,  which  is  a  nitrogen-containing  body, 
glucosmnin.^^ 

Simple  proteins  (e.  g.,  serum-globulin,  serum-albumin,  nucleo-albumin,  etc.) 
may,  when  in  solution  in  closed  cavities,  become  concentrated  through  absorption 
of  water  until  they  produce  the  phj^sical  appearance  of  "colloid."  Probably  the 
colloid  contents  of  dilated  renal  tubules,  cavities  in  various  mesoblastic  tumors, 
etc.,  are  produced  in  this  way. 

MUCOID  DEGENERATION 

Mucin,  in  its  typical  form,  is  a  compound  protein,  consisting  of  a 
protein  radical  and  a  conjugated  sulphuric  acid  which  contains  a 
nitrogenous  sugar.  Hence,  when  boiled  with  acids,  mucin  yields  a 
substance  reducing  Fchling's  solution.  Mucin  is  acid  in  reaction, 
probably  because  of  the  presence  of  the  sulphuric  acid  and,  therefore, 
is  characterized  microchemically  by  staining  with  basic  dyes.  It  is 
readily  dissolved  in  very  weak  alkahne  solutions,  is  precipitated  by 

68  Virchow's  Arch..  1906  (185),  416;  von  Sinner,  ibid.,  1915  (219),  279. 
«9Zangerle,  Munch,  med.  Woch.,  1900  (47),  414. 


MUCOID  DEGENERATION  431 

acetic  acid,  and  its  physical  properties  when  in  solution  are  quite 
characteristic.  The  term  mucin,  however,  probably  covers  a  number 
of  related  but  distinct  bodies.  Some,  such  as  the  pseudotnucins,  are 
readily  distinguished  by  not  being  precipitated  by  acetic  acid,  and 
by  being  alkaline  in  reaction;  others  yield  reducing  substances  with- 
out previous  decomposition  with  acids  (paramucin);  while  even  among 
the  "true"  mucins  certain  differences  in  solubility  exist. ^^  The  studies 
of  Levene^^  indicate  that  the  non-protein  radicals  of  mucins  are  of 
two  sorts:  One,  chondroitin-sulphuric  acid,  contains  the  nitrogenous 
hexose,  chondrosamine,  and  is  found  in  cartilage,  tendons,  aorta  and 
sclera;  the  other,  mucoitin-sulphuric  acid,  has  as  its  carbohydrate 
chitosamine,  and  is  found  in  gastric  and  umbilical  cord  mucin,  vitreous 
humor,  cornea  and  ovarian  cysts. 

In  the  mammalian  body  we  find  mucin  occurring  in  two  chief  lo- 
calities: (1)  as  a  product  of  secretion  of  epithehal  cells;  (2)  in  the 
interstices  of  connective  tissue,  especially  of  tendons. ^^  (The  resem- 
blance of  synovial  fluid  to  mucin  is  more  physical  than  chemical.) 
There  is  also  evidence  that  mucin  or  a  related  body  constitutes  the 
cement  substance  between  all  the  body-cells.  Corresponding  to  these 
two  chief  sources  of  mucin  we  find  mucoid  degeneration  occurring  as 
distinct  processes  in  mucous  membranes  (or  tissues  derived  therefrom) 
and  in  connective  tissue. 

Epithelial  Mucin. — As  epithehal  mucin  represents  a  distinct 
product  of  specialized  cells,  it  is  questionable  if  the  ordinary  applica- 
tion of  the  term  degeneration  in  the  sense  of  the  conversion  of  cell- 
protoplasm  into  mucin,  is  correct.  Certainly  the  mucin  formation  of 
catarrhal  inflammation  is  merely  an  excess  of  a  normal  secretion, 
and  the  degenerative  changes  that  may  be  present  in  the  epithelial 
cells  are  produced  by  the  cause  of  the  inflammation,  and  are  not 
dependent  upon  mucin  formation.  Even  in  the  extreme  example  of 
mucoid  degeneration  seen  in  carcinomas  derived  from  mucous  mem- 
branes (the  so-called  "colloid  cancers"),  the  epithelial  degeneration 
is  not  necessarily  to  be  interpreted  as  a  conversion  of  cell-cytoplasm 
into  mucin,  but  is  largely  due  to  the  pressure  of  secreted  mucin  upon 
the  cells  within  the  confined  spaces  of  the  tumor.  The  mucin  in  these 
forms  of  mucoid  degeneration  is  chemically  the  same  as  the  normal 
mucin  coming  from  the  same  source,  but  mixed  with  larger  or  smaller 
quantities  of  other  proteins  derived  from  cell  degeneration  or  from 

'"  For  special  consideration  see  Cutter  and  Gies,  Amer.  Jour.  Phvsiol.,  1901 
(6),  155. 

'"■  Jour.  Biol.  Chem.,  1918  (36),  105. 

'^  Schade  (Zeit.  exp.  Path.,  1913  (14),  23)  says  that  the  long  controversy 
concerning  the  intercellular  substance  of  mammalian  connective  tissue  is  settled 
by  the  work  of  Lier  (Ledermarkt-Collegium,  Frankfurt,  1909,  p.  321),  who  found 
it  to  be  a  mucin  similar  to  that  of  tendon  or  umbilical  cord.  Its  behavior  in 
edema  supports  this  observation.  That  there  are  some  chemical  similarities  in 
the  protein  moiety  of  epithelial  and  tendon  mucin  is  indicated  by  their  immuno- 
logical inter-reactions  (Elliott,  Jour.  Infect.  Dis.,  1914  (15),  501). 


432  RETROGRESSIVE  CHANGES 

vascular  exudates,  and  we  do  not  yet  know  certainly  the  chemical 
character  of  the  secretion  of  normal  mucous  membranes."  (The 
stringy,  mucin-like  substance  seen  in  some  purulent  exudates  is  prob- 
ably composed  largely  of  nucleoproteins  and  nucleo-albumins  derived 
from  the  degenerating  leucocytes^  and  is  not  true  mucin.) 

Connective=tissue  Mucin. — Excessive  formation  of  connective- 
tissue  mucin  is  observed  most  characteristically  in  myxedema  {q.  v.), 
but  may  also  occur  in  connective  tissues  that  are  poorly  nourished  or 
otherwise  slightly  injured;  it  is  seen  particularly  in  the  connective 
tissues  surrounding  the  epithelial  elements  in  adenomas  and  carcino- 
mas. In  the  walls  of  large  blood  vessels  there  is  a  mucoid  connective 
tissue,  rich  in  mucin,  which  may  be  increased  in  arterio-sclerosis 
(Bjorling).^^  Connective-tissue  tumors  (^myxosarcoma,  myxofibroma, 
or  myxoma)  may  also  show  a  great  quantity  of  mucinous  intercellular 
substance,  but  many  of  the  so-called  myxomas  are  in  reality  merely 
edematous  j&bromas  or  polypoid  tumors,  in  which  the  resemblance  to 
true  myxoma  is  largely  structural  rather  than  chemical.  This  form 
of  mucoid  degeneration  seems  to  be  merely  a  reversion  to  the  fetal  type 
of  connective  tissue,  which  is  characterized,  as  in  the  umbilical  cord, 
by  an  excessive  accumulation  of  a  mucin-containing  fluid  intercellular 
substance,  and  a  paucity  of  collagenous  fibrillar  structure.  Appar- 
ently, when  connective  tissue  reverts  to  an  embryonal  type,  either 
from  intrinsic  causes  (tumor  formation),  or  when  the  nourishment 
is  insufficient,  or  possibly  when  the  normal  stimulus  to  cell  growth  is 
absent  (myxedema),  the  mucoid  characteristics  of  fetal  tissue  reappear. 

The  presence  of  mucin  in  the  tissues  seems  to  cause  no  reaction, 
and  its  absorption  causes  no  harm.  Rabbits  that  I  injected  with 
large  quantities  of  pure  tendon  mucin  almost  daily  for  two  to  four 
months,  showed  absolutely  no  deleterious  effects,  either  locally  or  con- 
stitutionally. Some  of  the  French  authors^^  claim  that  mucin  pos- 
sesses a  slight  bactericidal  power.  On  the  other  hand,  Rettger^^  and 
others  have  found  an  apparently  typical  mucin  produced  by  certain 
varieties  of  bacteria. 

GLYCOGEN  IN  PATHOLOGICAL  PROCESSES" 

It  seems  probable  that  all,  or  nearly  all,  cells  contain  larger  or 
smaller  quantities  of  glycogen,  but  it  may  be  insufficient  in  amount 
to  be  detected  either  microscopically  or  chemicall3^  Glj^cogen  seems 
to  be  formed  within  the  cells  from  the  sugar  of  the  blood,  through  a 
process  of  dehydration  and  polymerization,  and  to  be  reconverted 
whenever  necessary  into  sugar,  by  a  reverse  process  of  hydrolysis.     It 

^^  See  Lopcz-Suarez,  Biochcm.  Zeit.,  1913  (56),  167. 
^*  Virchow's  Archiv.,  1911  (205),  71. 

"ArloiiiK.  Coinpt.  Rend.  Soo.  Biol.,  1902  (54),  306,  and  1901  (53),  1117. 
^«  Jour.  Med.  Research,  1903  (10),  101. 

"  Bibliography  by  Gierke,  Ziegler's  Beitr.,  1905  (37),  502,  and  Ergebnisso 
Pathol.,  1907,  XI  (2),  871;  Klestadt,  ibid.,  1911,  XYU),  349. 


GLYCOaENIC  IXFIl/rh'ATION  433 

is  quite  possible  that  both  of  these  processes  represent  merely  the 
reversible  action  of  an  intracellular  enzyme,  but  this  has  not  been 
estal)li8lio(l.  We  do  know,  however,  that  soon  after  death  the  intra- 
cellular glycogen  is  rapidly  converted  into  dextrose.^" 

Properties  of  Glycogen. — Glycogen  is  frequently  called  an  "animal  starch," 
having  the  same  f^eiieral  composition  as  the  starciies  (('bIIk.Oo)^,  and  apparently, 
like  the  starches,  it  represents  a  relatively  insolul)le  resting  stage  of  sugar  in 
the  course  of  metabolism.  It  is  readily  soluble  in  water,  forming  an  opalescent, 
colloidal  solution,  and,  therefore,  has  no  efTect  on  osmotic  pressure,  and  it  is  not 
difTusible."^  Because  of  its  solubility  and  the  rapidity  with  which  postmortem 
change  to  dextrose  occurs,  specimens  that  are  to  be  examined  microscopically  for 
glycogen  must  be  hardened  while  very  fresh  in  strong  alcohol,  in  which  glycogen 
is  insoluble.*"  One  of  the  most  characteristic  reactions  is  the  port-wine  color 
given  by  glycogen  when  treated  with  iodin;  this  reaction  may  be  applied  micro- 
scojncally,  solution  of  the  glycogen  being  avoided  by  having  the  iodin  dissolved  in 
a  solution  of  gum  arable  or  in  glycerol.  Salivary  ptyalin  rapidly  converts  gly- 
cogen into  glucose,  and  this  reaction  may  also  be  used  microscopically  to  prove 
that  suspected  granules  are  glycogen.  However,  failure  to  find  glycogen  micro- 
chemically  does  not  alwaj's  mean  its  absence  from  a  tissue.*' 

Physiological   Occurrence 

According  to  Gierke,  the  normal  glycogen  of  cells  resembles  fat  in  that  part  of 
it  disappears  during  starvation,  while  the  rest  cannot  be  removed  in  this  way  and 
probably  is  something  more  than  a  reserve  food-stuff.  In  distribution  glycogen 
somewhat  resembles  fat,  being  abundant  in  the  liver*'^  and  muscles,  but  Gierke 
considers  that  the  microscopic  evidence  of  the  quantity  of  glycogen  present  in  the 
cell  agrees  better  with  the  results  of  actual  chemical  analysis  than  is  the  case  with 
fat.  Ilusk,*^  however,  finds  only  a  general  agreement,  with  marked  exceptions. 
Neither  iodin  nor  Best's  carmin  stain  are  absolutely  specific  for  glycogen,  but 
Gierke  believes  that  we  may  safely  consider  a  substance  as  glj^cogen  when  it  is 
homogeneous,  rather  easily  soluble  in  water  and  more  so  in  saliva,  gives  the  usual 
iodin  reaction,  and  stains  bright  red  with  Best's  carmin  solution.*^  With  these 
controls,  the  microscopic  findings  were  found  to  agree  closely  with  the  results  of 
direct  chemical  analysis,  and  glycogen  was  found  microscopically  visible  in  muscle, 
liver,  lung,  heart,  uterus,  and  skin  (but  not  in  the  brain,*^  where  it  may  be  demon- 
strated chemically  in  minute  quantities). 

Glycogen  is  commonly  said  to  be  especially  abundant  in  fetal  tissues,  but  it  is 
not  present  in  all  fetal  cells, *^  nor  is  it  always  most  abundant  in  the  most  rapidly 
growing  tissues.  Although  both  fat  and  glj'^cogen  are  quite  abundant  in  fetal 
muscle  and  liver  tissues,  the  liver  of  early  embryos  does  not  contain  either.*' 
Invertebrates  and  the  lower  vertebrates  have  more  than  the  higher  forms.  In 
mammalian  adults  the  liver  and  muscle  contain  the  most  glycogen,  cartilage 

'* Literature  concerning  physiology  of  glvcogen  bv  Pflliger,  Pfliiger's  Arch., 
1903  (96),  398;  and  Cremer,  Ergeb.  der  Physiol.,  1902  (1.  Abt.  1),  803. 

'^  See  Gatin-Gruzewska,  Pfluger's  Arch..  1904  (103),  282. 

*°  According  to  Helman  (Cent.  f.  inn.  Med.,  1902  (23),  1017),  glycogen  may  be 
found  in  specimens  preserved  in  alcohol  as  long  as  fifteen  years. 

*i  Bleibtreu  and  Kato,  Pfluger's  Arch.,  1909  (127),  118. 

*^  In  the  livers  of  two  executed  criminals  Garnier  (Comp.  Rend.  Soc.  Biol., 
1906  (60),  125)  found  respectively  4  per  cent,  and  2.79  per  cent,  of  glycogen. 

»3  Univ.  of  California  Publ.,  Pathol.,  1912  (2),  83. 

**  Concerning  staining  methods  see  Ivlestadt,  loc.  cil.'''' 

**  Mav  be  present  in  fetal  nervous  tissues.  (Gage,  Jour.  Comp.  Xeurol.,  1917 
(27),  451). 

«8  See  Glinke,  Biol.  Zeit.,  Moskau,  1911  (2),  1. 

"  Adamof!  (Zeit.  f.  Biol.,  1905  (46),  288)  contests  the  idea  that  the  amount 
of  glycogen  is  in  direct  relation  to  growth  energy;  see  also  Mendel  and  Leaven- 
worth (Amer.  Jour.  Physiol.,  1907  (20),  117),  who  found  no  particular  abundance 
in  the  tissues  of  the  fetal  pig. 
28 


434  RETROGRESSIVE  CHANGES 

standing  next,  and  it  is  also  present  in  squamous  epithelium  (particularly  the  mid- 
dle layers),  especially  that  of  the  vagina  (Wiegmann),  but  not  in  slightly  stratified 
(cornea),  transitional,  or  cylindrical  epithelium.  Normal  human  kidneys  do  not 
seem  to  show  glycogen,  but  it  may  be  present  in  the  kidneys  of  mice,  rabbits,  and 
cats.  There  is  considerable  in  the  heart  muscle.*^  The  amount  in  different  skele- 
tal muscles  varies,^^  usually  being  especially  abundant  in  the  diaphragm.  Gly- 
cogen is  most  abundant  in  the  uterus  at  the  time  of  child-birth,  and  is  abundant  in 
the  placenta;  but  it  is  also  present  in  the  uterus  and  tubes  independent  of  preg- 
nancy.^" After  pancreas  extirpation,  Fichera^^  observed  a  disappearance  of  all 
visible  glycogen,  except  a  little  in  the  cartilage  and  stratified  epitheliuni ;  hence  he 
considers  the  glycogen-content  as  a  function  of  cell  nourishment.  Fat  and  gly- 
cogen often  occur  together,  although  one  may  be  present  without  the  other  (Gierke). 
Presumably  the  failure  to  find  glycogen  in  certain  cells  depends  rather  on  a 
failure  of  technic  than  on  a  total  absence  of  glycogen. 

There  has  been  some  diversity  of  opinion  as  to  whether  glycogen  occurs  as 
granules  in  the  living  cell,  or  whether  the  granules  are  formed  from  a  homogeneous 
substance  by  hardening  fluids.  In  view  of  the  clear-cut,  definite  spaces  it  may 
leave  in  cells  when  dissolved  out,  glycogen  probably  occurs  as  granules,  especially 
when  present  in  abnormally  large  quantities.  Ervin^^  believes  that  glycogen,  like 
fat,  may  exist  within  the  cells  so  finely  divided  that  it  cannot  be  stained  bj''  glycogen 
stains.  The  studies  of  Arnold  have  shown  that  in  many  cells  the  glycogen  takes  on 
a  definite  structure  in  close  relation  to  the  plasmosomes.  It  has  been  suggested 
that  the  intra-epithelial  hyaline  bodies  (Russell's  fuchsin  bodies)  are  glycogen, 
which  idea  is  probably  not  correct.  Habershon  and  others  have  suggested  that 
eosinophile  granules  are  either  glycogen  or  related  to  it.  The  presence  of  glycogen 
in  the  cells  seems  to  cause  no  injury  to  the  cytoplasm,  and  if  it  again  disappears, 
the  cells  become  quite  normal. ^^  Even  the  nuclei  may  contain  granules  of 
glycogen  without  evident  permanent  injury. 

Pathological  Occurrence 

According  to  the  results  obtained  by  Fichera  and  Gierke,  it  seems 
probable  that  glycogen  accumulation  is  produced  under  the  same 
conditions  as  are  fatty  changes,  i.  e.,  when  oxidation  is  locall}^  or 
generally  impaired.  Fat  and  glycogen  are,  therefore,  often  found 
together  in  the  margins  of  infarcts  and  of  tubercles,  in  passive  con- 
gestion of  the  liver,  and  in  heart  muscle  with  fatty  changes  due  to 
severe  anemia.  The  glycogen,  being  more  labile,  seems  to  disappear 
early  when  the  cells  become  necrotic,  and  hence  glycogen  is  not  pres- 
ent in  older  necrotic  areas  where  the  fat  still  persists.  (This  proba- 
bly accounts  for  the  frequently  repeated  statement  that  glycogen  and 
fat  do  not  occur  together.)  Ervin^-  believes  that  glycogen  is  impor- 
tant in  holding  intracellular  fats  emulsionized,  and  hence  in  its  ab- 
sence in  diabetes  the  fats  become  visible  as  fatty  degeneration — hence 
the  inverse  ratio  of  glycogen  and  fat.  Whether  the  glycogen  can  be 
transformed  into  fat,  perhaps  forming  an  intermediary  stage  in  a  trans- 
formation of  protein  into  fat,  has  not  been  determined,  but  there 

88  Berblinger,  Ziegler's  Beitr.,  1912  (53),  155. 
8M.ipska-Mlodowski,  Beitr.  path.  Anat.,  1917  (64),  18. 

90  McAllister,  Jour.  Obs.  Gyn.  Brit.  Emp.,  1913  (34),  91. 

91  Ziegler's  Beitr.,  1904  (30),  273,  literature. 

92  Jour.  Lab.  Chn.  Med.,  1919  (5),  14(). 

''Yet  Teissier  (Compt.  Rend.  Soc.  Biol.,  1900  (52),  790)  believes  the  amount 
normally  present  in  the  liver  is  strongly  bactericidal,  and  in  a  later  publication 
(ibid.,  1902  (54),  1098)  considers  that  it  is  toxic  to  liver-cells.  ^^Vndelstadt 
(Cent.  f.  Bact.,  Abt.  1,  1903  (34),  831)  found  that  under  certain  conditions  gly- 
cogen impedes  hoinoly.sis  by  normal  serum.  • 


GLYCOGENIC  INFILTRATION  435 

seems  to  be  little  doubt  that  it  is  infiltrated  from  outside  the  cell, 
and  not  formed  directly  from  degenerated  protein.  It  seems  to  be 
deposited  only  in  cells  that  are  still  living,  although  it  can  become 
split  up  in  dead  cells.  All  cells,  but  especially  muscle-cells  and 
leucocytes,  seem  able  to  lay  up  glycogen  in  visible  amounts  under  cer- 
tain conditions.  In  inflamed  areas  glycogen  is  found  in  both  ti-ssue- 
cells  and-  leucocytes,  but  not  in  cells  showing  nuclear  degeneration 
(Best,  Gierke).  In  pneumonia  the  leucocytes  of  the  exudate,  and 
to  a  less  extent  the  alveolar  epithelium,  contain  glycogen  as  well  as 
fat.  In  tubercles  glycogen  is  found  in  the  cells  which  contain  ba- 
cilli, and  it  is  generally  present  in  the  epithelioid  cells,  rarely  in  giant 
cells,  not  at  all  in  lymphoid  cells  or  in  the  necrotic  elements  (De- 
vaux).  Liver  glycogen  is  altered  most  in  poisoning,  being  reduced 
by  phosphorus,  arsenic,  chloroform,  HgCU,  and  many  other  poisons; 
the  amount  is  reduced  when  death  from  any  cause  is  slow,  or  when 
putrefaction  has  occurred,  but  it  is  increased  in  carbon  monoxide 
poisoning  (Alassari).^^  In  rabbits,  at  least,  it  is  deposited  in  the  liver 
first  about  the  central  vein,  and  in  fasting  animals  it  disappears  first 
from  the  periphery. ^^  It  seems  to  have  a  marked  protective  effect  in 
phosphorus  poisoning. ^^ 

Glycogen  in  Tumors. — Glycogen  has  been  observed  frequently 
in  tumors.  Brault  believed  the  quantity  an  index  of  rate  of  growth, 
on  the  principle  that  glycogen  appears  most  abundantly  in  embryonal 
tissues,  and  therefore  in  tumors  the  amount  of  glycogen  should  agree 
with  the  degree  to  which  the  cells  have  gone  back  to  the  embryonic 
tj'pe.  Lubarsch  considered  that  only  tissues  normally  containing 
glj^cogen  give  rise  to  glycogen-containing  tumors.  Gierke  could  cor- 
roborate neither  of  these  ideas,  and  considers  that  glycogen  appears  in 
tumors  under  exactly  the  same  conditions  in  which  it  appears  in  other 
tissues;  i.  e.,  when  cell  nutrition  and  oxidation  are  impaired.  Ap- 
parently, however,  both  the  embryonic  origin  and  local  retrogressive 
changes  determine  the  deposition  of  glycogen  in  tumors.  Glycogen 
is  particularly  abundant  in  squamous  epithelium  of  epitheliomas  that 
have  gone  on  to  hornification;^'^''  in  testicular  tumors,  hyperneph- 
romas, parathyroid  tumors  (Langhans),^^  endotheliomas,  chondromas, 
and  mj^omas,  and  it  also  occurs  in  the  connective  tissues  surrounding 
tumors.  Of  1544  tumors  of  all  sorts  examined  by  Lubarsch, ^^  447 
(or  29  per  cent.)  contained  glycogen  microscopically;  fibromas,  oste- 
omas, gliomas,  hemangiomas  were  always  free  from  glycogen;  and 
lipomas  and  lymphangiomas  nearly  always.     Adenomas  are  almost 

"   Gaz.  degli  Ospedali,  1906  (27),  537. 

"  Ishimori,  Biochem.  Zeit.,  1913  (48),  332. 

9«  See  Simonds,  Arch.  Int.  Med.,  1919  (23),  362. 

■^^^  In  mouse  tumors  Haaland  found  glycogen  only  in  squamous  cell  carcinoma, 
and  in  the  connective  tissue  surrounding  other  tumors  (Jour.  Path,  and  Bact., 
1908  (12),  439). 

^'    Virchow's  Arch.,  1907  (189),  138. 

"ss  Virchow's  Arch.,  1906  (183),  188. 


436  RETROGRESSIVE  CHANGES 

equally  free  from  glycogen  (two  positive  in  260  specimens),  while  it  was 
constant  in  teratomas,  rhabdomyomas,  hypernephromas,  and  chorio- 
epitheliomas.  Fifty  and  seven-tenths  per  cent,  of  the  sarcomas  and 
43.6  per  cent,  of  the  carcinomas  showed  glycogen,  most  abundant  in 
squamous-cell  epitheliomas;  columnar-celled  carcinomas  contain  gly- 
cogen much  less  often,  and  it  is  always  absent  in  "colloid  cancers." 

Animal  parasites,  in  common  with  other  invertebrates,  usually 
show  abundant  quantities  of  glycogen.  ^^  It  has  been  found  in  pro- 
tozoa, as  well  as  in  all  varieties  of  intestinal  worms.  According  to 
Barfurth,  nematodes  in  glycogen-free  animals  may  contain  glj'cogen. 
The  glycogen  is  found  chiefly  in  the  connective  tissues  of  the  intestinal 
parasites,  but  in  some  of  the  nematodes  it  occurs  chiefly  in  the  sexual 
organs  and  muscle-cells.  The  walls  of  the  hydatid  cysts  contain  much 
glycogen,  which  is,  perhaps,  related  to  the  usual  presence  of  sugar  in 
their  contents.  If  Habershon's  contention  is  correct,  that  eosinophile 
granules  are  related  to  glycogen,  we  may  have  here  an  explanation 
of  the  occurrence  of  eosinophilia  in  infection  with  animal  parasites. 
(See  also  "Animal  Parasites,"  Chap,  v.) 

Glycogen  in  Leucocytes. — The  occurrence  of  glycogen  in  the 
blood  has  aroused  much  interest,  particularly  in  relation  to  its  diag- 
nostic value.  Many  leucocytes  contain  granules  that  stain  with  iodin, 
and  although  it  is  possible  that  these  are  not  all  granules  of  glycogen, 
yet,  for  the  most  part,  they  probably  represent  this  substance  in 
excessive  quantities.  The  granules  are  observed  chiefly  in  the  poly- 
morphonuclear neutrophiles,  but  seldom  in  large  and  small  mononu- 
clear cells  and  eosinophiles.'  Occasional  granules  are  also  found  free 
(or  perhaps  contained  in  blood-platelets)  in  all  blood,  whether  normal 
or  pathological. 2  Hirschberg^  states  that  normal  animals  of  all 
species  have  leucocytes  giving  an  iodin  reaction  for  glycogen  if  proper 
technic  is  used,  but  which  is  not  obtained  by  the  ordinary  iodin-gum 
solution  method  unless  the  glycogen  is  rendered  abnormally  insolu- 
ble by  toxic  injury;  this  is  an  explanation  for  the  relationship  of 
iodophilia  and  infections.  According  to  Wolff-Eisner  the  leucocytes 
in  myeloid  leukemia  contain  no  glycogen  granules.  It  does  not  seem 
to  be  settled  whether  the  glycogen  is  taken  on  by  the  leucocytes  at 
the  place  of  pathological  lesion,  or  in  the  bone-marrow  under  the  in- 
fluence of  circulating  poisons,  or  both.  Habershon  states  that  from 
1  to  16  per  cent,  of  all  leucocytes  normally  contain  glycogen  granules, 

^^  Elaborate  treatise  on  occurrence  of  glycogen  in  lower  animals  by  Barfurth, 
Arch,  mikros.  Anat.,  1885  (25),  269;  also  liusch,  Arch,  internat.  physiol.,  1905 
(3),  49;  Brault  and  Loeper,  Jour.  Phys.  et  Path.  Gen.,  1904  (6),  295  and  720. 

1  See  Bond,  Brit.  Med.  Jour.,  Feb.  3,  1917. 

=^  Literature— Locke  and  Cabot,  Jour.  Med.  Research,  1902  (7),  25;  Locke, 
Boston  Med.  and  Surg.  Jour.,  1902  (147),  289;  Reich.  Bcitr.  klin.  Chir.,  1904  (42), 
277;  Kiittner,  Arch.  klin.  Chir.,  1904  (73),  438;  Gulland,  Brit.  Med.  Jour.,  1904 
(i),  880;  Habershon,  Jour.  Path,  and  Bact.,  1900  (11),  95;  Wolff,  Zeit.  klin.  Med. 
1904  (51),  407. 

3  Virchow's  Arch.,  1908  (194),  367. 


GLYCOGENIC  INFILTRATION  437 

and  Wolff  believes  that  the  glycogen  seen  in  leucocytes  represents 
normal  glycogen  made  insoluble  through  injury.  This  may  explain 
why  the  leucocytes  in  an  infected  area  may  give  iodin  reactions  when 
the  leucocytes  in  the  circulating  blood  do  not. 

Locke  gives  the  occurrence  of  this  abnormal  iodin  staining  of  the 
leucocytes  (termed  iodophilia)  as  follows:  "Septic  conditions  of  all 
kinds,  including  septicemia,  abscesses,  and  local  sepsis  (except  in  the 
earliest  stages),  appendicitis  accompanied  by  abscess  formation  or  per- 
itonitis, general  peritonitis,  empyema,  pneumonia,  pyonephrosis,  sal- 
pingitis with  severe  inflammation  or  abscess  formation,  tonsillitis, 
gonorrheal  arthritis,  and  hernia  or  acute  intestinal  obstruction  where 
the  bowel  has  become  gangrenous,  have  invariably  given  a  positive 
iodophilia,  and  by  its  absence  all  these  cases  can  be  ruled  out  in  diag- 
nosis. In  other  words,  no  septic  condition  of  any  severity  can  be 
present  without  a  positive  reaction.  Furthermore,  the  disappearance 
of  the  glycogen  granules  in  the  leucocytes  in  from  twenty-four  to 
forty-eight  hours  following  crisis  with  frank  resolution  in  pneumonia, 
and  the  thorough  drainage  of  pus  in  septic  cases,  is  of  considerable 
importance."  Clinical  experience,  however,  seems  not  to  have  ac- 
corded any  constant  significance  to  iodophilia.* 

In  exudates  glycogen  is  found  in  the  leucocytes  as  long  as  they 
retain  their  vitality,  but  disappears  soon  after  retrogressive  changes 
begin;  hence  it  is  not  usually  present  in  old  sterile  pus.  Loeper^ 
made  quantitative  estimates  of  the  glycogen  in  exudates,  finding  from 
0.59-0.62  gram  per  Liter  in  cellular  pneumococcus  pleural  effusion, 
0.25  gm.  in  cellular  tuberculous  effusion,  but  only  traces  in  serous 
tuberculous  effusion  and  in  an  old  tuberculous  pyothorax.  A  pneu- 
monic lung  contained  0.85  gm.  of  glj^cogen  per  kilo,  and  traces  were 
found  in  pneumonic  sputum  and  in  the  contents  of  tuberculous  cavi- 
ties. It  is  very  abundant  in  tuberculous  sputum,  as  much  as  2  to  3  per 
cent,  in  advanced  stages,  but  absent  in  bronchial  catarrh;  in  pneu- 
monia 0.05  per  cent,  was  found,  in  putrid  bronchitis  0.25  per  cent. 
(Pozzilli).  When  glycogen  solution  (1  per  cent.)  is  injected  into  the 
peritoneal  cavity,  the  endothelial  cells  and  invading  leucocytes  be- 
come loaded  with  glycogen  granules. 

Glycogenic  Infiltration  in  Diabetes. —  Although  in  diabetes 
the  chief  normal  storehouses  of  glycogen,  the  hver  and  muscles,  are 
either  poor  in  or  free  from  glycogen,  yet  in  other  tissues  in  diabetes 
the  most  marked  accumulations  of  glycogen  are  found,  the  granules 
frequently  fusing  in  the  cells  into  droplets  larger  than  the  nucleus. 
When  dissolved  out  in  ordinary  microscopic  preparations,  the  clear 
round  space  left  is  exactly  hke  the  space  left  by  a  fat-droplet,  except 
that  the  margins  show  a  tendency  to  take  the  basic  stain  for  some 
unknown  reason.  In  even  the  most  extreme  cases,  however,  the  nucleus 

*  See  Bernicot,  Jour.  Path,  and  Bact.,  190G  (11),' 304. 
»  Arch.  M6d.  Exp.,  1902  (14),  576. 


438  RETROGRESSIVE  CHANGES 

is  well  preserved  although  it,  too,  may  contain  large  masses  of  glycogen, 
in  which  case  there  is  no  glycogen  in  the  cytoplasm.''  Gl3'cogen  is 
found  particularly  in  the  epithelium  of  Henle's  tubules,"  in  heart 
muscle,  and  in  the  leucocytes.  Fiitterer  describes  masses  of  glj'cogen 
in  the  cerebral  capillaries,  resembling  an  embolic  process;  it  is  also 
present  in  the  tissues  of  the  eye.^  Experimental  diabetes  (pancreas 
extirpation,  piqure)  produces  a  marked  glycogenic  infiltration.^  We 
cannot  yet  change  van  Noorden's  statement:  "We  lack  the  biological 
explanation  as  to  why  certain  cells  retain  the  capacity  to  store  glj'co- 
gen  and  even  exert  it  more  actively  than  before,  whilst  the  proper 
organs  for  the  storage  of  glycogen  have  lost  it." 

*  Askanazy  and  Hubschmann,  Cent.  f.  Path.,  1907  (18),  041. 
'  See  Fahr,  Cent.  f.  Path.,  1911  (22),  945. 

*  Shimagawora,  Klin.  Monatsbl.  Augenheilk.,  1911  (12),  G82. 
9  Huber  and  MacLeod,  Amer.  Jour.  Physioh,  1917  (42),  019. 


CHAPTER  XVII 

CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

CALCIFICATION' 

Pathological  calcifioation  occurs  in  two  forms:  one  is  a  precipita- 
tion of  calcium  in  secretions  and  excretions  of  the  body;  the  other 
is  the  deposition  of  calcium  salts  in  the  tissues  themselves.  The 
former,  which  includes  not  only  concretions  in  general,  but  probably 
also  the  deposition  of  calcium  salts  in  the  cells  and  tubules  of  the 
kidney,-  both  in  disease  and  in  experimental  calcification  after  cer- 
tain poisonings,  is  readily  enough  explained  in  most  instances  by  rec- 
ognizable alterations  in  the  composition  of  the  secretions,  which  lead 
to  simple  chemical  precipitations.  With  this  form  we  shall  deal  in 
the  subsequent  consideration  of  concretions,  but,  in  referring  to  calci- 
fication, shall  indicate  only  depositions  from  the  blood  directly  into  the 
tissues.^ 

Relation  of  Calcification  to  Ossification. — In  normal  ossification  we  have  to 
deal  with  the  accumulation  of  lime  salts  within  the  stroma  or  cells  of  a  tissue 
that  has  usually  undergone  certain  preparatory  changes  in  the  way  of  formation 
of  a  more  or  less  homogeneous  ground  substance,  but  has  not  suffered  a  total  loss 
of  vitality,  although  vitality  is  possibly  decreased.  Pathological  calcification  is 
similar,  in  so  far  as  we  have  to  deal  with  deposition  of  quite  the  same  salts  in  tis- 
sues that  have  suffered  either  total  or  partial  loss  of  vitality,  and  which  very 
frequently  indeed  are  hyaline.  What  appear  to  be  essential  differences  are  these : 
(1)  In  calcification  the  lime  salts  always  remain  in  clumps  and  masses,  often 
fusing  to  greater  or  less  degree,  but  never  with  the  diffuse  even  permeation  of  tissue 
seen  in  ossification.  (2)  All  the  cells  within  a  calcified  area,  if  not  dead  at  the 
beginning  of  the  process,  eventually  disappear  for  the  most  part,  and  we  have 
sooner  or  later  a  perfectly  inert  mass,  practically  a  foreign  body,  instead  of  a 
specialized  tissue  as  in  ossification.  (3)  Ossification  is  accomplished  only  in 
varieties  of  connective  tissue,  but  calcification  may  involve  any  sort  of  cell  or 
tissue  provided  it  is  degenerated  sufficiently.  Furthermore,  anj^  area  of  calcification 
is  likely  to  be  replaced  by  bone,  no  matter  what  tissue  may  be  involved;  ap- 
parently the  presence  of  calcium  salt  deposits  in  any  part  of  the  body  can  stimu- 
late the  connective  tissues  to  form  bone,* but  in  the  absence  of  calcium  salts  even 
the  cells  which  are  normally  osteogenic  will  not  form  bone. 

1  Literature  and  resum6:  Pfaundler,  Jahrb.  f.  Kinderheilk.,  1904  (60),  123; 
Wells,  Jour.  Med.  Research,  1906  (14),  491,  and  Arch.  Int.  Med.,  1911  (7),  721; 
Hofmeister,  Ergebnisse  Phvsiol.,  1910  (9),  429;  Schultze,  Ergebnisse  Pathol., 
1910,  XIV  (2),  706. 

2  See  Wells,  Holmes  and  Henry,  Jour.  Med.  Research,  1911  (25),  373. 

^  Normally  the  calcium  content  of  the  blood  is  quite  constant,  about  9-11  mg. 
per  100  c.c.  serum,  and  the  quantit}'  is  not  modified  by  most  diseases,  except 
nephritis  in  which  the  serum  calcium  is  reduced;  also  in  eclampsia,  tetany  and 
jaundice.     (Halverson,  Mohlcr  and  Bergcim,  Jour.  Biol.  Chem.,  1917  (32),  171.) 

*  See  Nicholson  (Jour.  Path.  Bact.,  1917  (21),  287)  concerning  heterologous 
ossification. 

439 


440      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

Composition  of  the  Deposits  in  Calcification.^ — The  composi- 
tion of  the  inorganic  salts  in  calcified  areas  in  the  body  seems  to  be 
practically  the  same,  if  not  identical,  whether  the  salts  are  laid  down 
under  normal  conditions  (ossification)  or  under  pathological  condi- 
tions. With  the  blood  continually  passing  between  the  bones  and 
the  calcified  areas,  the  composition  of  the  two  must  inevitably  become 
similar  or  identical.  This  may  be  shown  by  a  table  giving  the  pro- 
portion of  inorganic  salts  found  by  analysis  of  normal  bone,  and  the 
proportion  found  in  calcified  materials.^ 


Mg3(P04)2 


CaCOs 


Cai(P04)j 


Pathological  Calcification 

Bovine  tuberculosis 

Bovine  tuberculosis 

Bovine  tuberculosis 

Bovine  tuberculosis  (softened  gland) 

Human  tuberculosis 

Calcified  nodule  in  thyroid .  .  . 

Thrombus,  human 

Normal  Ossification 

Human  bone  (Zalesky) 

Human  bone  (Carnot) 

Human  bone  (Carnot) 

Ox  bone  (Zalesky) 

Ox  bone  (Carnot) 


0.S4 

12.8 

0.9 

13.1 

1.2 

11.7 

1.5 

7.6 

1.2 

10.1 

0.85 

13.4 

1.1 

11.9 

1.04 

±12.8 

1.57 

10.1 

1.75 

9.2 

1.02 

1.53 

11.9 

85.9 
85.4 
86.4 
90.6 
87.8 
85.4 
86.5 


83.8 
87.4 
87.8 
86.1 
85.7 


Iron  may  be  present  in  pathological  calcification,  and,  according 
to  Gierke,^  in  the  fetus  the  entire  skeleton  contains  iron  as  far  as  it 
has  calcified,  most  at  the  points  of  active  ossification.  This  statement 
has  been  questioned  by  Hiick  and  others,  who  believe  that  most  of 
the  iron  demonstrable  in  normal  ossification  is  the  result  of  an  arti- 
fact, for  calcium  deposits  seem  to  have  a  great  afiinity  for  iron.  Be- 
cause of  this,  pathological  calcium  deposits  take  up  iron  from  old 
hemorrhages  in  the  vicinity,  and  so  in  many  areas  where  there  have 

^  MacCordick  (Lancet,  Oct.  18,  1913)  has  advanced  the  interesting  hypothesis 
that  calcific  deposits  during  life  exist  mostlj^  as  soft  masses,  like  unset  mortar. 
Only  when  sufficient  accumulation  of  CO2  occurs,  as  after  death,  or  in  the  center 
of  large  areas  of  low  vitality,  such  as  fibroids,  do  the  deposits  become  hardened; 
e.g.,  in  a  gangrenous  leg  the  calcified  vessels  are  stiff  and  brittle,  while  higher 
up  in  the  living  tissues  they  are  soft  and  pliable.  This  would  explain  why  we  do 
not  more  often  observe  fractures  of  calcified  arteries.  As  yet  this  hypotliesis  has 
not  received  the  critical  tests  its  importance  deserves.  If  true  it  will  explain 
the  cases  of  extensive  calcification  of  the  pericardium  in  which  the  heart  is  so 
encased  that  function  would  seem  impossible  if  the  deposit  were  rigid  during 
life.  (Sec  Trans.  Chicago,  Pathol.  Society,  1911  (8),  109,  for  consideration  of 
pericardial  calcification.)  However,  Klotz  (Jour.  Med.  Res.,  1916  (34).  495) 
has  questioned  the  correctness  of  MacCordick's  views  on  the  basis  of  the  occa- 
sional occurrence  of  fractures  of  calcified  arteries,  but  without  experimental  evi- 
dence contradicting  MacCordick. 

«  Wells,  loc.  cit. 

'  Virchow's  Arch.,  1902  (167),  318. 


PATHOLOGICAL   CALCIFICATION  441 

been  hemorrhages,  especially  in  the  vicinity  of  elastic  tissue,  th(!re 
occur  actual  "calcium-iron"  incrustations."  S.  Ehrlich^  states  that 
elastic  fibers  in  the  vicinity  of  hemorrhages  take  up  the  iron-contain- 
ing derivative  of  the  blood-pigment,  and  this  acts  as  a  mordant  for 
subsequent  calcium  deposition.  Analysis  of  similar  deposits  in  a 
syphilitic  spleen  by  Gettler^"  showed  the  presence  of  large  amounts  of 
silicates  as  well  as  calcium  and  iron.  Potassium  was  much  less  than 
in  normal  spleen  tissue.  The  presence  of  iron  in  normal  ossification 
is  supported  by  Sumita'^  and  Eliasscheff.*^  In  the  so-called  iron-lime 
lung  Gigon**  found  but  a  trace  of  calcium  and  much  sodium  and 
potassium. 

Structure  of  Calcified  Areas. — As  before  mentioned,  in  calcifi- 
cation there  is  not  the  same  uniform  infiltration  of  the  ground  sub- 
stance with  lime  salts  that  occurs  in  bone,  yet  the  calcified  area  is 
possessed  of  a  ground  substance  of  organic  material  which  does  not 
dissolve  in  weak  acids  that  remove  the  salts.  There  is  no  definite  ratio 
between  the  lime  salts  and  this  albuminoid  matrix,  however.  At 
first  the  salts  occur  in  granules,  which  may  become  fused  to  a  greater 
or  less  degree.  It  has  been  thought  by  some  that  the  deposition  occurs 
in  the  form  of  "calcospherites." 

These  are  small  calcareous  bodies,  usually  of  concentric  structure,  which  were 
first  described  by  Harting.  They  appear  to  occur  widely  distributed  in  normal 
tissues,  both  animal  and  plant,  and  seem  to  be  the  result  of  the  formation  of 
insoluble  calcium  salts  in  the  presence  of  colloidal  substances,  just  as  urinary 
and  other  concretions  are  formed  about  an  organic  nucleus.  If  calcium  chloride 
and  soluble  carbonates  are  allowed  to  combine  very  slowly  to  form  calcium  car- 
bonate in  a  solution  of  egg-albumen,  these  or  indistinguishable  bodies  are  formed, 
which  on  being  dissolved  are  found  to  possess  an  organic  stroma  that  exhibits  a 
marked  affinity  for  any  pigmentary  substance  that  may  be  present.  Apparently, 
when  the  proper  concentration  exists,  the  salts  in  crj'stallizing  hold  between  the 
crystals  the  albuminous  substances  by  which  they  are  surrounded.  Dastre  and 
Morat  believe  that  the  substratum  is  lecithin,  which  others  have  found  occupying 
a  similar  place  in  prostatic  concretions.  Calcospherites  have  been  found  in  tumors, 
in  cystic  cavities,  and  in  bodies  with  beginning  decomposition.  It  may  be  men- 
tioned in  passing  that  Littlejohn'^  observed  the  abundant  formation  of  calcium 
phosphate  crystals  in  bodies  that  had  been  immersed  for  some  time  in  sea  water. 
Oliver  has  found  calcospherites  in  the  tissues  of  a  cancer  of  the  breast.  Pettit'^ 
found  calcospherites  in  a  sarcoma  of  the  maxilla,  presenting  insensible  transi- 
tions into  the  substance  of  the  osseous  tissue,  and  he  suggests  the  possibility  that 
the  calcospherite  formation  may  be  related  to  the  formation  of  bone.  It  seems, 
however,  that  they  are  probably  more  closely  related  to  the  formation  of  the 
shells  of  invertebrates,  which  are  largely  composed  of  carbonates  in  crystalline 
structure  with  an  organic  ground  substance  between  them,  and  very  little  phos- 
phate indeed. 

« See  Gigon,  Ziegler's  Beitr.,  1912  (55),  46;  Sprunt,  Jour.  Exp.  Med.,   1911 
(14),  59;  Klotz,  Johns  Hop.  Hosp.  Bull.;  1916  (27),  363. 
"  Cent.  f.  Pathol.,  1906  (17),  177. 

'» Symmers,  Gettler,  Johnson,  Surg.,  Gyn.  Obst.,  1919,  (28),  58. 
"  Virchow's  Arch.,  1910  (200),  220. 
12  Ziegler's  Beitr.,  1911  (50),  143. 
>3  Edinburgh  Med.  Jour.,  1903  (13),  127. 
1*  Arch.  d.  Anat.  Micros.,  1897  (1),  107. 


442      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

Occurrence  of  Pathological  Calcification 

As  far  as  we  know,  calcification  seldom  occurs  in  normal  tissue, 
except  in  the  formation  of  bone.  Often  the  infiltrated  tissue  is  com- 
pletely dead,  as  in  infarcts,  organic  foreign  bodies,  caseous  areas,  and 
particularly  in  old  inspissated  collections  of  pus.  It  may  be  said 
that  any  area  of  dead  tissue  that  is  not  infected,  and  that  is  so  large 
or  so  situated  that  it  cannot  be  absorbed,  will  probably  become  infil- 
trated with  lime  salts.  Most  frequently  calcified,  next  to  totally 
necrotic  tissues,  are  masses  of  scar-tissue  that  have  become  hyaline  sub- 
sequent to  the  shutting  off  of  circulation  in  the  scar  by  contraction 
of  the  tissue  about  the  vessels.  Elastic  tissue  also  seems  prone  to  an 
early  calcification,  and  it  is  not  uncommon  to  see  the  elastic  laminae 
of  small  arteries  calcified  in  an  apparently  selective  manner.  A  pe- 
culiar form  of  calcification  is  that  frequently  found  in  ganglion-cells 
of  the  brain  which  have  become  degenerated  or  necrotic,  particularly 
in  the  vicinity  of  old  hemorrhages;  the  cells  become  infiltrated  with 
lime  salts  until  a  complete  cast  of  the  cell,  with  dendrites  and  axis- 
cylinder  well  impregnated,  is  formed.  The  calcification  of  renal  epi- 
thelium obtained  experimentally  by  temporary  ligation  of  the  renal 
vessels  or  by  the  administration  of  certain  poisons,  is  more  closely 
related  to  the  formation  of  ordinary  urinary  concretions  than  to  tissue 
calcification,  the  calcium  being  present  as  the  phosphate  only.^^  Cal- 
cification of  epithelial  cells  does  occur,  however,  and  seems  to  be  pre- 
ceded by  hyaline  changes,  in  which  hyaline  substance  the  calcium  is 
later  deposited,  as  in  epithelial  pearls,  for  example. 

Metastatic  Calcification. — What  is  perhaps  the  only  exception 
to  the  rule  that  some  form  of  tissue  degeneration  is  required  before  cal- 
cification occurs,  is  the  "metastatic  calcification"  of  Virchow.^^  In 
conditions  with  much  destruction  of  bone,  as  osteomalacia,  caries, 
osteosarcoma,  etc.,  deposits  of  lime  salts  have  been  found  distributed 
diffusely  in  various  organs,  particularly  in  the  lungs  and  stomach. 
As  much  as  13.38  per  cent,  of  the  dry  weight  of  the  lung  and  12.15 
per  cent,  of  the  kidney  have  been  found  as  CaO  in  such  a  case.*^ 
As  there  is  no  evidence  that  these  organs  have  been  the  site  of  any  dif- 
fuse tissue  necrobiosis  before  the  calcification  occurred,  it  seems  prob- 
able that  the  deposits  have  been  made  in  practically  or  quite  normal 
organs,  because  of  oversaturation  of  the  tissue  fluids  by  calcium  salts. 
The  fact  that  the  lung  and  stomach,  and  also  to  a  less  degree  the  kid- 
ney, are  picked  out,  suggests  that  the  calcification  is  related  to  the 
fact  that  in  these  same  organs  we  have  the  excretion  of  acids  into 
their  cavities,  which  leaves  the  fluids  in  the  substance  of  the  organs 
correspondingly  alkaline,   and   an   increase   in  the  alkalinity  of  the 

iMour.  Med.  Iles^earch,  1911  (25),  373. 

"Virchow's  Arch.,  1855  (8),  103;  review  l)V  Kockol,  Dent.  Arch.  klin.  Med., 
1899  (U4),  332.  Bil)lioKraphy  and  review  by  Wells,  Arch.  int.  Med.,  1915  (15), 
574. 

"  Virchow's  Arch.,  1909  (197),  112. 


PATHOLOGICAL  CALCIFICATION  443 

fluids  makes  the  calcium  salts  decidedly  less  soluble.  In  the  stomach 
the  calcium  deposits  arc  limited  to  the  interglandular  tissue  about 
the  upixn-  portion  of  the  shmds  of  the  fundus,  exactly  corresponding 
to  the  parietal  cells  which  are  supposed  to  secrete  the  acid.  Pre- 
sumabl}^,  under  normal  conditions,  the  amount  of  calcium  in  the 
blood  is  too  slight  to  be  thrown  down  in  this  way,  but  when  oversat- 
urated  because  of  the  calcium  absorption  in  the  skeleton,  precipita- 
tion occurs  in  the  parts  of  the  bodj^  where  the  alkalinity  of  the  blood 
or  tissue  fluids  is  greatest,  or  the  CO2  concentration  least.  There  also 
occurs  a  true  metastatic  calcification  in  the  large  arteries,  pulmonary 
veins,  and  beneath  the  endocardium  of  the  left  side  of  the  heart;  that 
is,  always  in  the  places  where  the  blood  contains  the  least  CO?.  This 
fact  supports  the  hypothesis  that  the  CO?  is  an  important  factor  in  the 
solution  of  calcium  salts  in  the  blood,  and  that  when  there  is  an  over- 
saturation  with  calcium  it  is  deposited  where  the  CO2  is  least  abun- 
dant. When  the  amount  of  calcium  in  the  blood  is  increased  by 
injecting  or  feeding  calcium  salts,  depositions  of  calcium  salts  may  take 
place  in  injured  tissues, ^^  or  even  in  normal  tissues,  as  in  Tanaka's 
experiments.^^  Extensive  calcification  may  take  place  in  the  lungs 
without  any  evident  bone  disintegration,  nor  yet  nephritis  which  has 
been  thought  at  times  to  lead  to  enough  calcium  retention  to  account 
for  metastatic  calcification  (Harbitz).^°  A  few  cases  of  extensive 
subcutaneous  calcification  of  unknown  etiology  have  been  described, 
but  their  relation  to  metastatic  calcification  is  doubtful,  as  they  seem 
to  be  localized  deposits. ^^ 

Some  have  attempted  to  include  the  calcification  of  the  vessels  and 
other  tissues  in  old  age  in  the  metastatic  calcifications,  ascribing  the 
origin  of  the  salts  to  the  senile  absorption  of  bone,  but  senile  calcifica- 
tion is  probably  dependent  rather  upon  the  extensive  hyaline  degenera- 
tion of  the  connective  tissues  that  occurs  in  the  senile  scleroses,-^  a 
change  which  seems  to  be  more  physical  than  chemical.-^ 

Chemistry  of  the  Process  of  Calcification 

In  analyzing  the  etiological  factors  in  the  production  of  pathologi- 
cal calcification  for  the  purpose  of  determining  the  chemical  changes 
that  occur  in  the  process,  we  have  the  following  facts  upon  which  to 
base  the  consideration: 

(1)  The  calcium  salts  must  come  from  the  blood,  where  they  are 

18  See  Thayer  and  Hazen,  Jour.  Exp.  Med.,  1907  (9),  1. 

i^Biochem.  Zeit.,  1911  (35),  113;  (38),  285;  see  also  Katase,  Beitr.  path. 
Anat.,  1914  (57),  516. 

2«  Norsk  Mag.  Laeg.,  1917  (78),  1129. 

21  See  Mosbacher,  Deut.  Arch.  klin.  Med.,  1918  (128),  107. 

^^  Under  the  name  of  "calcium  gout,"  M.  B.  Schmidt  has  described  a  case  with 
generalized  deposition  of  calcium  in  other  tissues  than  those  usually  affected  in 
metastatic  calcification  (Deut.  med.  Woch.,  1913  (39),  59). 

^^  See  analyses  of  elastin  from  calcified  and  normal  aortas  by  Ameseder,  Zeit. 
physiol.  Choin.,  1913  (85),  324. 


444      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

held  in  solution  or  in  suspension  by  the  proteins,  either  as  the  car- 
bonate and  phosphate  themselves,  or  as  calcium-ion-protein  com- 
pounds, or  perhaps  both.  This  suspension  or  solution  is  an  unstable 
condition,  possibly  only  because  of  the  extremely  small  proportion  of 
calcium  in  the  plasma  (about  1  :  10,000),  and,  therefore,  capable  of 
being  overthrown  by  increased  alkalinity  of  the  blood,  changes  in  the 
proteins  or  CO2  content,  or  changes  in  the  quantity  or  composition 
of  the  calcium  salts.  It  is  probable,  from  the  work  of  Barille,  that 
the  calcium  of  the  blood  exists  as  a  soluble  complex  double  salt,  tri- 
basic  calcium-carbon-phosphate  (P208Ca2H2:  2C02(,C03H)2Ca),  this 
compound  being  possible  because  of  an  excess  of  CO2. 

(2)  Retrogressive  changes  in  the  tissues  are  a  sine  qua  non  except 
in  metastatic  calcification.  Hyaline  degeneration,  the  chemical 
nature  of  wliich  is  not  understood,  is  a  very  favorable  condition,  as 
also  is  necrosis  when  absorption  is  deficient. 

(3)  In  the  areas  that  are  to  become  calcified  the  circulation  is 
very  feeble,  the  blood  plasma  seeping  through  the  tissue  as  through 
any  dead  foreign  substance  of  similar  structure,  without  the  presence 
of  red  corpuscles  to  permit  of  oxidative  changes. 

We  may,  therefore,  imagine  that  the  deposition  of  calcium  salts 
in  such  areas  of  tissue  degeneration  depends  upon  one  or  more  of  the 
following  conditions: 

(1)  Increased  alkalinity  or  decreased  CO?  in  the  degenerating 
tissues,  causing  precipitation  of  the  inorganic  salts  in  the  fluids  seep- 
ing slowly  through  them. 

(2)  Utihzation  of  the  protein  of  the  fluids  by  the  starved  tissues 
so  completely,  because  of  its  slow  passage  through  them,  that  the 
calcium  cannot  be  held  longer  in  solution. 

(3)  The  formation  within  the  degenerated  area  of  a  substance  or 
substances  having  a  special  affinity  for  calcium. 

(4)  Production  of  a  physical  condition  favoring  the  local  absorp- 
tion of  salts,  the  least  soluble  salts  accumulating  in  excess. 

The  first  of  these  conditions  seems  to  come  into  play  especially  in 
metastatic  calcification,  already  discussed.  We  have  no  evidence  that 
in  degenerating  tissues,  much  less  in  normal  ossification,  there  is  an 
alkahne  reaction  developed;  but  rather  the  contrary,  an  acid  reaction 
is  more  usual.  But,  as  explained  below,  decrease  in  the  CO2  content 
in  calcifying  tissues,  especially  when  combined  with  other  changes, 
may  be  of  importance. 

Lichtwitz24  especially  has  laid  emphasis  on  the  possible  part  played 
by  changes  in  the  proteins  in  inducing  calcification.  He  advances 
the  idea  that  precipitation  of  the  colloids  in  the  degenerated  area, 
as  in  caseation,  decreases  the  amount  of  crystalloids  which  can  be 
held  in  solution,  wherefore  the  least  soluble  salts,  those  of  calcium, 
arc  precipitated;  by  laws  of  osmotic  pressure  more  calcium  in  solu- 

"  Deut.-med.  Woch.,  1910  (36),  704. 


PATHOLOGICAL  CALCIFICATION  445 

tion  will  then  enter  to  establish  equilibrium,  be  precipitated,  and 
make  way  for  more  calcium,  until  the  amount  of  deposit  prevents 
further  osmotic  diffusion.  Although  suggestive  in  regard  to  patho- 
logical calcification,  and  probably  of  importance  in  the  formation 
of  concretions,  this  conception  is  difficult  toapi)ly  to  normal  ossification; 
also  in  pathological  calcification  one  would  expect  precipitation  of 
calcium  to  occur  in  the  outermost  surface  of  the  degenerated  area, 
soon  leading  to  a  shell  of  inorganic  material  which  would  limit  the 
deposition. 

The  possibihty  of  the  formation  of  calcium-binding  substances 
within  the  degenerated  area  has  always  seemed  the  most  attractive, 
and  has  received  the  most  attention  by  investigators.  Of  the  special 
substances  that  might  be  present  in  such  areas  that  would  have  a 
high  affinity  for  calcium,  phosphoric  acid  usually  receives  first  con- 
sideration, since  it  is  as  phosphate  that  most  of  the  calcium  is  bound, 
and  also  since  the  possible  sources  of  phosphoric  acid  in  decomposed 
nucleoproteins  and  lecithin  are  so  obvious.  Less  considered  in  the 
past,  fatty  acids  offer  another  possibility,  especially  in  view  of  the 
fatty  degeneration  that  so  frequently  precedes  calcification.  Proteins 
might  also  be  formed  that  would  combine  calcium,  especially  dcutero- 
albumose,  which  Croftan^^  states  has  a  high  degree  of  affinity  for 
calcium,  and  which  would  be  present  in  areas  undergoing  autolysis. 

Formation  of  Calcium  Soaps. — In  favor  of  the  possibility  that 
the  calcium  is  first  bound  as  soaps  are  the  following  facts:  Calcifica- 
tion occurs  chiefly  in  places  where  fatty  degeneration  has  occurred, 
such  as  tubercles,  atheromatous  vessels,  etc.  In  fat  necrosis  fatty 
acids  are  formed,  which  soon  combine  wuth  calcium  to  form  calcium 
soaps.  Virchow  observed  calcification  in  the  form  of  soaps  in  a 
lipoma,  and  Jaeckle^®  found  that  a  calcifying  Lipoma  contained  29.5 
per  cent,  of  its  calcium  in  the  form  of  calcium  soaps.  Klotz-^  ob- 
tained staining  reactions  in  calcifying  tissues  that  suggested  the  pres- 
ence of  soaps,  which  he  also  extracted  by  solvents,  and  he  strongly 
urges,  as  the  first  step  in  the  formation  of  pathological  calcified 
masses,  that  the  calcium  is  first  laid  down  as  soaps,  afterward  under- 
going a  transformation  into  the  less  soluble  phosphate  and  carbonate. 
Fischler  and  Gross-*^  also  obtained  microchemical  reactions  for  soaps 
in  the  margins  of  infarcts  and  in  atheromatous  areas,  but  not  in 
caseous  areas;  they  therefore  consider  that  calciimi-soap  formation  is 
an  important  step  in  the  process  of  pathological  calcification,  but 
that  it  is  not  essential.  The  value  and  the  interpretation  of  the  his- 
tological evidence  of  the  participation  of  calcium  soaps  is,  however, 
open  to  question. 

=5  Jour,  of  Tuberculosis,  1903  (5),  220. 

2«Zeit.  phvsiol.  Chem.,  1902  (36),  53. 

"Jour.  Exper.  Med.,  1905  (7),  663;  1906  (8),  322. 

28Ziegler's  Beitr.,  1905  (7th  suppl.),  339. 


446      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

On  the  other  hand,  Wells, ^^  studying  large  quantities  of  material 
chemically,  found  at  most  doubtful  traces  of  calcium  soaps  in  calci- 
fying matter,  even  in  the  earliest  stages,  and  also  very  small  amounts 
of  other  soaps  or  fatty  acids,  and,  therefore,  questions  the  occurrence 
of  calcium  soaps  as  an  essential  step  in  calcification,  although  not 
doubting  that  under  certain  conditions  {e.  g.,  calcifying  lipomas,  fat 
necrosis)  this  may  occur.  In  calcification  at  all  stages  the  propor- 
tion of  calcium  carbonate  and  phosphate  was  found  quite  constant, 
and  exactly  the  same  as  in  normal  bone;  namely,  in  the  proportion 
expressed  by  the  formula  3(Ca3(P04)2:CaC03,  which  Hoppe-Seyler 
advanced  to  express  the  composition  of  the  salts  of  bone.  Hence  it 
seems  probable  that  there  are  no  essential  differences  between  the 
processes  of  ossification  and  pathological  calcification,^"  and  there 
seems  to  be  as  yet  no  reason  for  assuming  that  in  the  former  calcium 
soaps  constitute  an  essential  step  in  the  process. 

Phosphoric  Acid  in  Calcification. — It  has  generally  been  as- 
sumed that  in  normal  ossification  the  calcium  is  combined  by  phos- 
phoric acid,  which  probably  is  derived  from  the  cartilage  cells,  possibly 
through  autolysis  of  the  nucleoproteins  or  some  similar  process. ^^ 
Grandis  and  Mainini,^^  by  using  microchemical  methods,  thought 
that  they  found  evidence  that  the  phosphorus  of  ossifying  cartilage  is 
converted  from  an  organic  combination  into  an  inorganic  form  (P2O5), 
which  then  takes  up  calcium  from  the  blood.  The  methods  used  have 
been  questioned,  and  Pacchioni,^^  from  his  studies,  was  inclined  to  the 
opinion  that  the  calcium  entered  the  cartilage  already  combined  as 
phosphate.  Wells  implanted  into  the  abdominal  cavity  of  rabbits 
various  tissues  that  had  been  killed  and  sterilized  by  boiling,  and 
found  that  tissues  rich  in  nucleoproteins  showed  no  tendency  to  take 
up  calcium  in  greater  amounts  than  did  tissues  poor  in  nucleoproteins, 
which  result  speaks  against  the  idea  that  phosphoric  acid  derived 
from  nucleic  acid  combines  the  calcium.  On  the  other  hand,  im- 
planted dead  cartilage  soon  became  thoroughly  impregnated  with 
calcium  salts,  which  seemed  to  be  deposited  in  the  same  proportion 
as  to  carbonate  and  phosphate  as  in  bone. 

Physical  Absorption  of  Calcium  Salts. — As  there  could  be  no 
question  of  "vital  activity"  on  the  part  of  this  boiled  cartilage,  it 
seems  most  probable  that  there  exists  in  cartilage  a  specific  absorp- 
tion affinity  for  calcium  salts,  similar  to  the  absorption  affinity  that 
Hofmeister^^  observed  exhibited  by  other  organic  colloids   (gelatin 

29  See  review  in  Arch.  Int.  Med.,  1911  (7),  721. 

^^  Dyes  that  stain  the  bones  when  fed  to  living  animals  (madder)  also  stain 
pathological  calcific  deposits  (Macklin,  Anat.  Kecord,  1917  (ll),  387). 

^'  Hanes,  who  observed  that  the  i)hosi)hatids  disaj)pcar  from  the  liver  of  the 
developing  chick,  suggests  this  as  a  source  of  the  phosphoric  acid  required  for 
ossification  (.Jour.  Exper.  Med.,  1912  (16),  512). 

^2  Arch,  per  la  sci.  Med.  Torino,  1900  (24),  67. 

"  Jahrb.  f.  Kinderheilk.,  1902  (56),  327. 

"  Arch,  exper.  Path.  u.  Pharm..  1891  (28),  210. 


I 


OSTEn}rALAnA  447 

disks)  toward  various  crystalline  suhstaiiccs  in  solution.  It  is  of  sig- 
nificance that  the  substances  in  which  calcium  is  deposited  are,  in 
most  instances,  of  similar  physical  character,  being  homogeneous  and 
often  hyaline,  although  of  the  most  varied  chemical  composition;  in 
other  words,  they  agree  much  more  in  physical  than  in  chemical  struc- 
ture. Also  we  find  that  hyaline  tissues  with  an  affinity  for  calcium 
often  exhibit  a  similar  affinity  for  other  substances,  such  as  pigment  and 
iron.^^  Hofmeister  advances  the  hypothesis  that  when  the  cartilage 
or  other  matrix  becomes  saturated  with  calcium  salts,  any  decrease 
in  COo  content  of  the  solution  will  lead  to  a  precipitation  of  calcium 
salts,  thus  restoring  to  the  cartilage  its  power  of  absorbing  more 
calcium  salts  whenever  the  fluid  comes  to  it  with  a  higher  degree  of 
saturation  with  calcium  salts  and  CO2.  This  hypothesis  is  in  har- 
mony with  Barille's  observation  that  when  the  C02  is  reduced  the 
complex  carbon-phosphate  of  calcium  precipitates  a  mixture  of  car- 
bonate and  phosphate  in  the  same  proportions  as  found  in  bones  and 
calcific  deposits  generally.  The  fact  that  this  ratio  (10  to  15  per 
cent.  CaCOs  and  85  to  90  per  cent.  Ca?(P03)4),  is  found  in  all  stages 
of  calcification,  is  entirely  in  favor  of  the  above  hypothesis,  and 
opposed  to  the  idea  that  any  special  chemical  precipitant  formed  in 
the  calcifying  area  is  responsible  for  the  deposition  of  calcium.  Taken 
all  in  all,  the  evidence  seems  in  favor  of  the  view  that  normal  ossifica- 
tion and  pathological  calcification  (except  metastatic  calcification 
and  the  calcification  of  fat  necrosis  and  other  areas  of  necrotic  fat 
tissue)  depend  more  upon  physico-chemical  factors  and  variations  in 
CO2  concentration  than  upon  the  presence  of  chemical  precipitants 
in  the  tissues.  This  view  is  supported  by  the  observation  of  IMacklin^'' 
that  calcifying  and  ossifying  tissues  become  stained  alike  with  madder 
fed  during  their  formation,  through  the  deposition  of  stained  calcium 
salts  from  the  blood. 

Osteomalacia" 

In  this  condition  the  quantity  of  inorganic  salts  in  the  bone  is 
greatly  decreased,  while,  at  the  same  time,  their  place  is  taken  in  part 
by  new-formed  osteoid  tissue;  as  a  result,  the  proportion  of  the  weight 
of  the  bone  formed  by  inorganic  salts  is  reduced  to  as  lew  as  20  to 
40  per  cent.,  instead  of  being  from  56  to  60  per  cent.,  as  in  normal 
bone.  This  has  suggested  that  the  cause  of  the  disease  may  be  a 
solution  of  the  lime  salts  by  some  acid,  but  Levy^^  found  that  in  osteo- 
malacia the  proportion  of  calcium  carbonate  and  phosphate  in  the 

"  See  Sprunt,  Jour.  Exp.  Med.,  1911  (14),  59. 

'«  Jour.  Med.  Res.,  1917  (36),  493. 

"  See  also  review  in  Albu  and  Neuberg's  "Mineralstoffwechsel,"  Berlin.  1906, 
pp.  124^127;  bibliography  by  Zesas,  Cent.  Grenz.  Med.  u.  Chir.,  1907  (10),  801; 
full  discussion  by  McCrudden,  Arch.  Int.  Med.,  1910  (5),  596;  1912  (9),  273. 

38  Zeit.  physiol.  Chem.,  1894  (19),  239. 


448      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

bones  remains  constant,  as  also  does  the  proportion  of  calcium  and 
phosphoric  acid;  if  the  decalcification  occurred  through  solution  by 
lactic  or  other  acids,  he  argued,  the  carbonate  should  be  decomposed 
first, ^^  whei'eas  the  lime  salts  seem  to  be  taken  out  as  molecules  of 
calcium  carbonate-phosphate;  i.  e.,  in  the  same  proportion  as  they 
exist  in  the  bone.  On  the  other  hand,  it  has  been  found  in  Pawlow's 
laboratory  that  dogs  kept  for  long  periods  after  a  pancreatic  fistula 
has  been  established,  develop  a  condition  resembling  osteomalacia,^" 
which  would  seem  most  reasonably  explained  as  due  to  the  constant 
loss  of  alkali  in  the  pancreatic  juice.  Furthermore,  investigation 
of  Levy's  objection  to  the  acid  solution  theory  has  led  to  the  observa- 
tion that  when  mixtures  of  calcium  carbonate  and  phosphate  are  in 
colloids  they  are  dissolved  at  equal  rates. *^  Histologically,  absorption 
seems  to  depend  largely  upon  a  direct  eating  out  of  bone  tissue,  both 
organic  and  inorganic  substance,  by  osteoclasts  (Cohnheim),  followed 
by  a  formation  of  an  uncalcified  osteoid  tissue.  (Senile  osteoporosis 
differs  chiefly  in  that  no  new  osteoid  tissue  is  formed.)  According  to 
Dibbelt^^  when  osteomalacia  is  experimentally  induced  in  pregnant 
dogs  and  then  recovery  is  allowed  to  take  place,  the  decalcified  bone 
substance  present  in  the  active  stage  does  not  become  calcified,  but  is 
absorbed  and  replaced  by  new  bone. 

Studies  of  metabolism  in  osteomalacia  have  shown  a  loss  of  calcium 
by  the  body,  especially  in  the  urine,  as  shown  by  the  following  table 
given  by  Goldthwait  et  al.:^^ 


Limbeck 

Neumann 

Goldthwait 

CaO  in  urine  (gm.) 

CaO  in  feces 

1.773 
3.834 

3.859 
1.800 

Total  excreted 

5.607 
2.965 

11.65 
11.26 

5.66 

Total  in  food 

4.56 

Loss  of  CaO 

2.9G5 

0.39 

1.10 

McCrudden  also  found  a  considerable  retention  of  nitrogen  and 
sulphur,  which  may  be  retained  in  the  new-formed  osteoid  tissue; 
magnesium^^  is  also  retained,  probably  being  substituted  for  calcium 
in  the  bones.     It  is  known  that  when  magnesium  and  strontium  are 

^'  Goto  reports  that  in  experimental  HCl  acidosis  the  bones  lost  20  per  cent, 
of  their  CaCOa  without  appreciable  loss  of  phosphate  (Jour.  Biol.  Chenu,  1918 
(36),  .355). 

">  Babkin,  Zeit.  Stoflfwechsel,  1910  (11),  561;  Looser,  Vcrh.  Deut.  Patli.  Gcsell., 
1907  (11)    291. 

"  Kranz  and  Liesegang,  Deut.  Monat.  Zahnhcilk.,  1914,  p.  628. 

^2  Arbeit.  Path.  Inst.  Tubingen,  1911  (7),  559. 

*' Goldthwait,  Painter,  Osgood  and  McCrudden,  Amer.  Jour.  PhvsioL,  1905 
(14),  389. 

**  Corroborated  by  Cappezzuoli,  Biochem.  Zoit.,  1909  (16),  355. 


RICKETS  449 

given  to  growing  animals  they  will  partially  replace  the  calcium  in 
the  bones,'*''  while  it  is  said  by  Etienne''*^  that  excessive  feeding  of  calcium 
itself  leads  in  time  to  decalcification  of  the  bones.  Zuntz**^  found  the 
respiratory  metabolism  in  osteomalacia  within  normal  limits,  but 
tending  to  be  low;  protein  metabolism  shows  nothing  striking,  but 
there  is  a  high  excretion  of  phosphoric  acid  through  the  feces. 

Castration  of  women  with  osteomalacia  has  been  frequently,  but 
not  always,  followed  by  improvement  or  recovery,^**  and  Neumann, 
and  also  Goldthwait,  have  found  that  in  these  cases  the  calcium  loss  is 
replaced  by  a  marked  calcium  retention  after  the  operation.  What 
the  relation  of  the  ovaries  to  calcium  metabolism  or  to  osteomalacia 
may  be  has  not  yet  been  ascertained.  Scharfe^'-*  and  Bucura^"  both 
state  that  there  are  no  characteristic  or  constant  structural  altera- 
tions in  the  ovaries  in  osteomalacia  McCrudden^'  found  that  the 
improvement  in  calcium  metabolism  observed  after  castration  may 
be  but  temporary,  and  therefore  believes  that  the  primary  cause  of 
the  disease  does  not  lie  in  the  ovaries.  He  is  of  the  opinion  that  re- 
peated drains  on  the  calcium  of  the  bones,  incited  most  often  by  preg- 
nancy, occasionally  by  tumors,  sometimes  by  unknown  causes,  result 
in  an  excessive  reaction  to  the  stimuli,  so  that  eventually  the  losses 
become  too  great  to  be  made  up;  that  is,  osteomalacia  is  an  exaggera- 
tion of  a  normal  process  resulting  either  from  excessive  stimulation 
of  that  process,  or  a  failure  to  recover  when  the  stimulus  ceases.  The 
beneficial  effects  of  castration  are  probably  ascribable  chiefly  or  solely 
to  the  prevention  of  pregnancy.  Osteitis  deformans  seems  to  be  a 
localized  osteomalacia.  The  relation  of  the  adrenals  to  osteomalacia 
advocated  by  Bossi,^^  is  of  questionable  significance,  and  there  is  no 
definite  evidence  as  to  any  relation  of  exophthalmic  goiter^^  or  the  para- 
thyroids,^* although  hyperplasia  of  the  parathyroids  has  been  des- 
cribed.^^ 

RlCKETS^s 

As  wdth  osteomalacia,  chemical  studies  of  the  bones  in  rickets  have 
thrown  httle  light  upon  the  etiology  or  pathogenesis  of  this  condition. 

*5  See  Lehnerdt,  Zeit.  exp.  Med.,  1913  (1),  175. 

•»«  Jour.  Physiol,  et,  Path.,  1912  (14),  108. 

"  Arch.  f.  Gyn.,  1913  (99),  145. 

^»  Bibliography  by  Schnell,  Zeit.  Geb.  u.  Gyn.,  1913  (75),  178. 

*3  Cent.  f.  Gyn.,  1900  (24),  1216. 

="  Zeit.  f.  Heilk.,  1907  (28),  209. 

"  Amer.  Jour,  of  Physiol.,  1906  (17),  211. 

"  Zent.  f.  Gyn.,  1907  (31),  69  and  172. 

"  Tolot  and  Sarvonat,  Rev.  d.  Med.,  1906  (26),  445. 

"  Erdheim,  Cent.  med.  Wiss.,  1908  (46),  163. 

^5  Bauer,  Frankfurter  Zeit.  Pathol.,  1911  (7),  231. 

"  Complete  literature  and  full  discussion  by  Pfaundler,  Jahr.  f.  Kinderheilk., 
1904  (60),  123;  also  see  Albu  and  Neuberg,  " Mineralstoffwechsel,"  Berlin,  1906. 
pp.  119-124;  symposium  in  the  Verhandl.  Deut.  Path.  Gesellsch.,  1909  (13),  1. 
Metabolism  studies  by  Meyer,  Jahrb.  Kinderheilk.,  1913  (77),  28. 

29 


450      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

As  the  following  table  (taken  from  Vierordt^^)  shows,  there  is  a  marked 
deficiency  in  the  proportion  of  inorganic  salts  in  the  bones  in  rickets. 
The  proportion  of  the  different  salts  seems  to  be  quite  the  same  as  in 
normal  bone. 


Normal  bone 

of  a  two 

months  old 

child 

Rachitic  bones 

Tibia 

Ulna 

Femur 

Tibia 

Humerus 

Ribs      Vertebrae 

Inorganic  matter 

65.32 
34.68 

64.07 
35.93 

20.60 
79.40 

33.64 
66.36 

18.88 
81.12 

37.19 
62.91 

32.29 
67.71 

57.54 
1.03 
6.02 
0.73 

33.86 
0.82 

56.35 
1.00 
6.07 
1.65 

34.92- 
1.01 

14.78 
0.80 
3.00 
1.02 

72.20 
7.20 

26.94  1        ,.  .„ 

Magnesium  phosphate 

0.81/ 
4.88 
1.08 
60.14  1 
6.22/ 

2.66 
0.62 

81.22 

Collagen  (or  ossein) 

Fat             

More  modern  analyses^**  show  a  relative  increase  in  water  and 
magnesium,  with  a  persistence  of  the  normal  ratio  of  calcium  phosphate 
and  carbonate.  ^^  Cattaneo®°  finds  the  increase  in  magnesium  to  vary 
in  different  parts  of  the  skeleton,  being  greatest  in  the  ribs.  Aschen- 
heim  states  that  the  blood  of  cliildren  with  rickets  shows  greater 
variations  from  the  usual  CaO  content  (8-10  mg.  per  100  c.c.)  than  are 
found  in  normal  children,^'  which  is  not  corroborated  by  others."' 

As  an  essential  difference  from  osteomalacia  is  the  fact  that  in 
rickets  there  is  a  failure  on  the  part  of  the  osteoid  tissues  to  calcify, 
whereas  in  osteomalacia  absorption  of  calcified  tissue  takes  place  with 
subsequent  substitution  by  osteoid  tissue.  Furthermore,  in  rickets  the 
deficiency  in  calcium  is  said  to  be  present  only  in  the  bones, "^  whereas 
in  osteomalacia  the  soft  tissues  are  also  poor  in  lime  salts.  According 
to  SchmorP^  the  first  structural  abnormality  in  rickets  is  a  failure 
to  lay  on  calcium  by  small  islands  of  cartilage  in  the  zone  of 
preparatory  calcification. 

None  of  the  various  hypotheses  as  yet  advanced  to  explain  this 
defective  ossification  has  satisfactorily  accounted  for  all  the  observed 
facts.  That  a  deficiency  of  calcium  in  the  food  is  the  cause  of  rickets 
is  a  most  natural  assumption,  but  it  has  not  been  proved  that  this  is  the 
case.  Young  animals  fed  on  calcium-poor  foods  show,  naturally  enough, 
defective  development  of  the  bone,*''*  but  this  differs  essentially  from 

^^  Nothnagel's  System,  vol.  7,  part  ii,  p.  21. 

"  Gassmann,  Zcit.  physiol.  Chem.,  1910  (70),  IGl. 

^'  The  bones  and  muscles  in  Barlow's  disease  show  quite  the  same  deficiencj'' 
in  calcium  as  in  rickets  (Bahrdt  and  Edelstein,  Zeit.  Ivinderheilk.,il913  (9),  415). 

60  La  Pediatria,  VII,  497. 

8'  Jahrb.  Kinderheilk.,  1914  (79),  446. 

"2  There  is  a  decrease  in  the  calcium  of  the  muscles  according  to  Aschenheini 
and  Kaumheimer  (Monatschr.  f.  Kinderlieil.,  1911  (10),  435) 

"  Verhandl.  Deut.  Path.  Gesell.,  1905  (9),  248. 

6^  See  Weiser,  Biochcm.  Zeit.,  1914  (66),  95. 


RICKETS  451 

rickets  in  that  the  bone  formed  is  defective  chicfl}'  in  amount  rather 
than  in  quality  (Stoltzner),  Furthermore,  such  "pscudo-rachitic 
bone"  possesses  a  marked  affinity  for  calcium  salts,  and  takes  them  up 
as  soon  as  the}'  are  supplied  (Pfaundler).  As  the  blood  in  rickets 
contains  nearly  normal  amounts  of  calcium^^  it  seems  quite  certain 
that  calcium  starvacion  is  not  the  fundamental  trouble.  In  view  of 
the  fact  that  rickets  is  not  solely  a  disease  of  bone  tissue,  but  that 
all  the  various  important  viscera,  as  well  as  the  muscles  and  tendons, 
show  pathological  changes,  it  seems  most  reasonable  that  rickets 
should  be  looked  upon  as  a  constitutional  disease,  in  which  the  bone 
changes  are  prominent  chiefly  because  the  disease  occurs  at  a  time  when 
the  bone  tissue  is  most  actively  forming  and  when  the  other  organs 
are  relatively  quite  completely  developed.  Stdltzner,^''  finding  evidence 
that  rickets  does  not  depend  upon  either  lack  of  calcium  in  the  food  or 
deficient  absorption  of  calcium,  and  that  the  blood  in  rickets  is  of  nor- 
mal alkalinity,  looks  upon  the  failure  of  calcification  as  depending  upon 
an  abnormality  in  the  calcified  bone  tissue  itself."  He  finds  evidence 
of  a  preliminary  alteration  in  normal  osteoid  tissue  which  prepares  it 
to  take  the  salts  out  of  the  blood,  and  Pfaundler-^^  supports  this 
view,  suggesting  that  this  preparatory  change  in  the  osteoid  tissue 
may  depend  upon  autolysis,  which  is  perhaps  deficient  in  rickets. ^^ 

On  the  other  hand,  after  extensive  experimental  work,  Dibbelt^^ 
comes  to  the  conclusion  that  rickets  results  from  excessive  elimination 
of  calcium  into  the  intestine,  presumably  because  of  the  presence  of 
precipitating  substances  in  the  intestinal  contents,  such  as  P-Oo  from 
casein.  Agreeing  with  Dibbelt  that  the  excessive  elimination  of  cal- 
cium is  chiefly  through  the  feces,  Schabad^"  aftei'  equally  extensive 
investigations,  believes  .that  calcium  starvation  in  children,  from  defec- 
tive absorption,  may  cause  at  least  a  pseudo-rickets,  indistinguishable 
clinically  or  chemically  from  true  rickets.  But  the  fact  that  children 
with  rickets  show  nearly  normal  figures  for  blood  calcium  does  not 
agree  with  these  calcium  starvation  hypotheses. 

As  with  osteomalacia,  attempts  have  been  made  to  associate  with 
the  etiology  of  rickets  defects  in  the  ductless  glands,  especially  the 
adrenals,'"  thymus, ^^  and  parathyroids,'-  but  as  yet  without  convinc- 
ing evidence.  ^^     There  has  also  been  an  attempt  to  include  rickets 

"Howland  and  Marriott, '  Trans.  Assoc.  Amer.  Phys.,  1917  (32),  307. 

««  Jahrb.  f.  Kinderheilk.,  1899  (50),  268. 

^''  How  metallic  i)hosphorus  causes  growing  bones  to  laj'  on  increased  calcium 
is  an  unsolved  problem,  but  a  striking  fact.  (See  Phemister,  Jour.  Amer.  Med. 
Assoc,  1918  (70),  1737.) 

«8  See  also  Nathan,  Med.  News,  1904  (84),  391. 

*^  Articles  in  the  Arbeiten  a.  d.  Path.  Inst.  Tubingen,  Vols.  6  and  7;  also  ^'erh. 
Deut.  Path.  GeselL,  1910  (-14),  294;  Miinch.  nied.  Woch.,  1910  (57),  2121. 

'0  Arch.  f.  Kinderheilk.,  1909  (52),  47;  1910  (53),  381;  1911  (54),  83;  Fortschr. 
Med.,  1910  (28),  1057. 

'"■  Stoeltzner,  Verh.  Deut.  Path.  Ges.,  1909  (13),  20. 

^2  Erdheim  et  al,  Frankfurter  Zeit.  Path.,  1911  (7),  178. 

'^  Concerning  the  chemical  changes  of  osteogenesis  imperfecta  (congenital  fra- 
gility of  bones),  see  Schabad,  Zeit.  Kinderheilk.,  1914  (11),  230. 


452      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

among  the  diseases  that  depend  upon  specific  deficiencies  in  the  diet, 
especially  the  fat-soluble  "vitamines"  which  cod-liver  oil  supplies 
abundantly.  So  far,  however,  this  hypothesis  is  not  positively  estab- 
Jished.^^  (See  also  Rickets,  under  ''Deficiency  Diseases"  Chapter  xii.), 

CONCRETIONS 

All  pathological  concretions  appear  to  be  laid  down  according  to  a 
definite  law.  There  must  first  be  a  nucleus  of  some  substance  differ- 
ent from  the  substance  that  is  to  be  deposited,  and  which  is  most 
frequently  a  mass  of  desquamated  cells,  but  may  consist  of  clumped 
bacteria,  masses  of  mucus,  precipitated  proteins,  or  a  foreign  body  of 
almost  any  sort.  Upon  this  nucleus  substances  crystallize  out  of 
solution,  much  as  cane-sugar  crystallizes  on  a  string  to  form  rock 
candy,  but  with  the  important  exception  that  among  the  crystals  is 
usually  deposited  more  or  less  mucin  or  other  organic  substance,  which 
forms  a  framework  in  which  the  crystals  lie,  and  which  remains,  if  the 
crystals  are  dissolved  out,  as  a  more  or  less  perfect  skeleton  of  the 
concretion.  In  no  case  would  the  concretion  form  were  it  not  that 
the  solution  is  overcharged  with  some  substance,  but  not  infrequently 
it  is  the  presence  of  the  nucleus  that  leads  to  the  precipitation  of  the 
substance;  i.  e.,  the  nucleus  may  play  either  a  primary  or  a  secondary 
role.  With  few  exceptions,  the  dissolved  substance  is  deposited  in 
crystallme  form,  although  the  crystalline  structure  may  in  time  partly 
disappear  through  condensation  or  through  filling  of  the  interstices 
with  some  other  material.  Even  so  structureless  a  substance  as 
amyloid  may,  when  forming  concretions,  appear  in  a  crystalline  form 
(Ophiils).  The  structure  of  a  concretion  depends  upon  two  factors: 
The  crystals  tend  to  be  deposited  at  right  angles  to  the  surface,  and 
thus  give  a  radiating  structure;  but  the  rate  of  deposition  is  usually 
irregular,  and  during  the  periods  of  quiescence  the  surface  tends  to 
become  covered  with  mucin  or  other  organic  substances,  hence  we  also 
get  a  concentric,  laminated  structure.  Frequently  both  of  these  lines 
of  formation  are  easily  discerned,  but  either  one  or  the  other  may  be- 
come obscured. 

Concretions  consist,  therefore,  of  mixtures  of  colloids  and  crystal- 
loids deposited  from  solutions  of  the  same  character,  and  hence  the 
application  of  the  principles  of  colloidal  chemistry  throws  much 
light  on  the  conditions  of  their  formation."'''^  Colloidal  solutions 
hold  in  solution  greater  quantities  of  crystalloids  than  simple  solutions, 
for  the  reason  that  at  the  surface  of  each  colloidal  particle  there  is  a 
zone  in  which  the  crystalloids  are  more  concentrated  than  elsewhere, 
thus   permitting  more   crystalloids  to   be  dissolved  in   the    solvent 

""^  See  Paton,  Findlay  and  Watson,  Brit.  Med.  Jour.  Dec.  7,  1918;  Mellanbv* 
Lancet,  1919  (196),  407. 

^6  See  Schade,  Munch,  med.  Woch.,  1909  (5G),  3;  1911  (58),  723;  Zeit.  exp. 
Path.,  1910  (8),  92;  also  Lichtwitz,  Ergeb.  inn.  Med.,  1914  (13),  1;  also  his  mono- 
graph "Ueber  die  Bildung  der  Ilarn-  und  Gallensteine,"  Springer,  Berlin,  1914. 


BILIARY  CALCULI  453 

between  the  colloidal  particles.  On  the  other  hand,  the  concentra- 
tion of  the  crystalloids  on  the  surface  of  the  colhjidal  particles  causes 
the  colloids  to  serve  as  the  starting  point  of  precii)itation  whenever 
the  crystalloids  are  in  excess.  When  the  crystalloid  goes  out  of  solu- 
tion, therefore,  it  will  form  crystals  or  precipitates  which  are  most 
intimately  associated  with  the  colloids,  as  we  see  when  uric  acid 
crystallizes  out  of  urine,  taking  with  it  the  colloidal  pigments  by  which 
it  is  absorbed.  Or,  if  the  colloids  are  precipitated,  the  solvent  power 
of  the  solution  is  reduced,  and  the  crystalloids  will  deposit  in  intimate 
relation  to  the  colloids.  As  Schade  pointed  out,  if  a  colloid  precipi- 
tates in  an  irreversible  form  {e.  g.,  fibrin),  the  concretion  will  be  per- 
manent, as  with  ordinary  concretions,  but  if  the  colloid  precipitate  is 
reversible  the  mass  may  be  dissolved  again,  as  with  the  precipitate  of 
urates  in  the  tubules  of  the  infant's  kidney. 

Biliary    Calculi'^ 

As  may  be  judged  from  the  above  statements,  concretions  are  never 
composed  of  one  substance  in  a  pure  form,  but  usually  consist  of  a 
mixture  of  the  constituents  of  the  fluid  in  which  they  are  developed. 
This  is  particularly  true  of  gall-stones,  which  contain  in  greater  or 
less  quantities  several  or  all  of  the  constituents  of  the  bile.  While 
cholesterol  forms  the  greater  part  of  nearly  all  biliary  concretions,  and 
is  present  in  greater  or  less  amounts  in  all,  calcium  salis  of  the  bile- 
pigments  are  always  present;  usually  inorganic  salts  of  calcium  (car- 
bonate and  phosphate)  are  also  present,  as  well  as  small  amounts  of 
fats,  soaps,  lecithin,  mucus,  and  other  products,"  and  occasionally 
traces  of  copper, '^^  iron,  and  manganese. '^^  The  quantity  of  bile 
salts,  the  chief  constituent  of  the  bile,  is  usually  extremely  minute, 
apparently  only  so  much  as  may  percolate  into  the  crevices  of  the  con- 
cretion. However  many  stones  there  may  be  in  a  gall-bladder,  they 
usually  are  all  of  approximately  the  same  composition  and  structure. 

In  gall-stones  from  the  domestic  animals  the  proportion  of  inor- 
ganic salts  is  usually  much  higher  than  it  is  in  man. 

Naunyn  has  classified  gall-stones  according  to  their  composition, 
as  follows: 

1.  "Pure"  Cholesterol  Stones. — The  purity  is  only  relative,  since  even  the 
purest  alwaj'S  contain  some  pigment  as  well  as  a  stroma  and  a  nucleus;  but  the 
amount  of  cholesterol  may  reach  98  per  cent.,  and  is  usually  over  90  per  cent. 
Crystalline  structure  is  usually  well  marked,  while  stratification  is  slight.  The 
color  varies  from  nearl}^  pure  white  to  yellow,  or  even  brown  on  the  surface. 

2.  Laminated  Cholesterol  Stones. — These  consist  of  about  75-90  per  cent,  of 
cholesterol,  and  differ  from  the  preceding  form  in  containing  more  pigment,  which 

"8  Bibliography  by  Bacmeister,  Ergeb.  inn.  Med.,  1913  (11),  1. 

^^  Fischer  and  Rose  found  about  0.1  gm.  carotin  in  1280  grns.  gall  stones  from 
cattle.     (Zeit.  physiol.  Chem.,  1913  (88),  331.) 

'8 See  Mizokuchi,  Cent.  f.  Pathol.,  1912  (23),  337. 

"^Gall-stones  have  been  found  enclosing  droplets  of  mercury.  (Xaunyn, 
Frerichs.) 


454      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

is  deposited  in  layers  alternating  with  the  white  layers  of  cholesterol.  The  pig- 
ment here,  as  in  all  other  gall-stones,  consists  always  of  the  calcium  salts  of  the 
pigments — not  of  pure  bilirubin  and  biliverdin  themselves.  Considerable  cal- 
cium carbonate  is  also  usually  present,  particularly  in  the  green  layers  of  biliverdin- 
calcium. 

3.  Common  Gall-bladder  Stones. — The  composition  of  this  form  is  but  little 
different  from  the  above,  the  chief  difference  being  in  the  structure.  They  pre- 
sent externally  a  firmer  crust,  usually  distinctly  laminated;  in  the  center  is  a  softer 
pigmented  nucleus  which  frequently  shows  a  central  cavity  containing  fluid. 
Such  calculi  are  not  distinctly  crystalline  in  structure,  and  are  small,  seldom 
larger  than  a  cherry. 

4.  Mixed  Bilirubin-calcium  Calculi. — These  generally  occur  singly,  but  some- 
times in  groups  of  three  or  four,  and  are  of  large  size.  Although  the  chief  con- 
stituent is  bilirubin-calcium,  there  is  always  much  cholesterol,  often  over  25  per 
cent.  Copper  and  traces  of  iron  may  also  be  present.  Their  structure  is  lamin- 
ated, with  sometimes  a  crystalline  cholesterol  nucleus. 

5.  "Pure"  Bilirubin-calcium  Calculi. — In  addition  to  the  chief  constituent, 
bihver din-calcium,  hilifuscin  and  hilihumin^^  are  practically  always  present.  Bili- 
humin  is  at  times  the  chief  ingredient,  and  may  form  over  half  of  the  substance; 
hilicyanin  is  rarely  present.  There  is  always  some  cholesterol,  but  sometimes 
only  traces.  These  calculi  are  small,  from  the  size  of  a  grain  of  sand  to  that  of  a 
pea,  and  they  occur  in  two  distinct  forms.  One  form  is  of  wax-like  consistence; 
the  other  is  harder,  steel-gray  or  black  in  color,  with  a  metallic  luster.  Pure  bili- 
rubin and  biliverdin,  not  combined  with  calcium,  are  practically  never  present 
in  concretions. 

6.  Rarer  Forms. — (a)  Amorphous  and  incompletely  crystalline  cholesterol  gravel. 
Cholesterol  externally  giving  a  pearly  luster;  pigment  in  the  center. 

(b)  Calcareous  Stones. — Consist  chiefly  of  a  mixture  of  calcium  carbonate  and 
bilirubin-calcium.  Calcium  carbonate  may  occur  either  as  a  superficial  crust, 
or  as  small  masses  within  an  ordinary  calculus;  calcium  sulphate  and  phosphate 
occur  rarely  in  traces.  Stones  consisting  mainly  of  calcium  carbonate  are  ex- 
tremely rare  in  man,  but  more  frequent  in  cattle  and  other  herbivora,  in  which 
all  forms  of  concretions  contain  much  calcium,  either  combined  with  pigment 
or  as  carbonate  and  phosphate.  A  calcium  oxalate  gall-stone  has  also  been 
described.  ^^ 

(c)  Concretions  with  included  bodies,  and  conglomerate  stones. 

(d)  Casts  of  Bile-ducts. — Occur  particularly  in  cattle,  and  consist  chiefi}^  of 
bilirubin-calcium.     Rarely  and  imperfectly  formed  in  man. 

Aschoff  and  Bacmeister  differ  somewhat  from  Naunyn  as  to  the 
composition  of  gall-stones,  which  they  classify  as  follows: 

1.  Pure  cholesterol  stones. 

2.  Stratified  cholesterol-calcium  stones. 

3.  Cholesterol-pigment-calcium  stones. 

4.  Composite  stones,  composed  of  cholesterol  and  a  mantle  of  cho- 
lesterol and  calcium. 

5.  Bilirubin-calcium  stones,  usually  found  in  the  bile  passages  of  the 
liver. 

6.  The  very  rare  calcium  carbonate  stones. 

Formation  of  QaII=stones. — Until  quite  recently  our  views  con- 
cerning the  chemistry  and  pathology  of  the  formation  of  gall-stones 

*"  Biliverdin  differs  from  bilirubin  in  containing  one  more  atom  of  oxygen  in 
the  molecule,  and  it  is  easily  formed  from  bilirubin — even  exposure  to  air  will 
slowly  bring  about  the  oxidation.  Bilifusciit  is  a  still  more  oxidized  derivative — 
so  much  so  that  it  does  not  give  Cmeliii's  reaction  (with  HNO3+HNO2)  for  bile- 
l)igments.  Bilihumin  rei)resents  the  most  oxidized  of  these  products,  is  brown 
in  color,  and  is  the  chief  constituent  of  the  residue  left  after  treating  gall-stones 
with  ether,  alcohol,  and  chloroform  to  dissolve  out  the  cholesterol. 

**  Montlaur,  Bull.  sci.  i)harmacol.,  Vol.  IS,  p.  19. 


BILIARY  CALCULI  455 

were  dominated  by  the  observations  and  conclusions  of  .Naunyn*^ 
and  his  pupils.  Former  observers,  having  learned  that  bile  normally 
contains  cholesterol  (Hammarstcn  found  from  0.06-0.  IG  per  cent,  in 
human  bile),  sought  the  cause  of  gall-stones  in  either  an  increased 
elimination  of  cholesterol  by  the  liver,  or  a  decrease  in  the  power  of 
the  bile  to  hold  the  cholesterol  in  solution.  Thus  Frerichs,  finding 
that  the  presence  of  large  amounts  of  bile  salts  and  an  alkaline  re- 
action favored  the  solution  of  cholesterol,  imagined  that  a  diminu- 
tion of  either  bile  salts  or  alkalinity  led  to  the  precipitation  of  the 
cholesterol.  Naunyn  and  his  pupils,  however,  not  finding  that  the 
amount  of  cholesterol  present  in  the  bile  depends  upon  the  amount 
taken  in  the  food  or  the  amount  present  in  the  blood,  and  that  it 
did  not  vary  in  disease,  except  when  gall-stones  were  present,  con- 
cluded that  the  cholesterol  of  the  bile  is  neither  a  product  of  general 
metabolism  nor  a  specific  secretion-product  of  the  liver.  Finding 
that  pus  and  the  secretions  from  inflamed  mucous  membranes  (bron- 
chitis) contained  as  much  cholesterol  as  did  normal  bile,  and  often 
more,  they  concluded  that  the  chief  source  of  cholesterol  in  gall-stone 
formation  was  from  the  degenerating  and  desquamated  epithelial 
cells  of  the  gall-bladder  and  bile  tracts.  This  idea  was  supported  by 
the  large  amount  of  cholesterol  found  in  the  contents  of  gall-bladders 
shut  off  from  the  common  duct,  and  by  the  formaton  of  gall-stones 
in  such  isolated  gall-bladders.  Some  further  evidence  has  since  been 
brought  forward  in  favor  of  this  same  view,^^  but  others,  finding 
no  abundance  of  cholesterol  in  the  wall  of  the  gall-bladder  have  not 
accepted  this  origin.'*'* 

On  the  basis  of  Naunyn's  hypothesis  the  ordinary  steps  in  the  for- 
mation of  a  cholesterol  concretion  are  as  follows:  Some  injury  to  the 
mucous  membrane  of  the  bile  tracts  is  the  starting-point;  this  injury 
is  usually  produced  by  infection,  the  colon  and  typhoid  bacilli  being 
the  most  common  organisms  in  this  process. ^^  Through  the  degenera- 
tion of  the  epithelial  cells  an  excess  of  cholesterol  is  formed,  while  at 

*2  An  English  translation  of  this  classic  work,  by  A.  E.  Garrod,  has  been  pub- 
lished by  the  Sydenham  Society,  1896,  vol.  158. 

*^  Thus  Wakeman  (quoted  by  Herter,  Trans.  Congress  Amer.  Physicians,  1903 
(6),  158)  was  able  to  cause  an  increase  in  the  cholesterol  of  the  bile  in  the  gall- 
bladder of  dogs  by  injecting  into  it  HgClo,  phenol,  or  ricin.  At  first  the  choles- 
terol seems  to  be  contained  largely  in  the  degenerating  desquamated  cells.  Also 
the  interesting  case  of  a  cholesterol  calculus  in  a  pyosalpinx,  described  by  Thies 
(Arb.  Path.  Inst.  Tubingen,  1908  (6),  422),  shows  the  possibility  of  an  inflamma- 
tory origin  for  such  concretions,  and  independent  of  bile. 

■«^  Aschoff,  Miinch.  med.  Woch.,  1906  (53),  1847  and  1913  (60),  1753;  Aschoff 
and  Bacmeister,  "Cholelithiasis,"  Gustav  Fischer,  Jena,  1909;  Laroche  and 
Flandin,  Compt.  Rend.  Soc.  Biol.,  1912  (72),  660. 

8^  See  Cushing  (Johns  Hopkins  Hosp.  Bull.,  1899  (10),  166),  who  produced 
gall-stones  experimentally  by  injecting  typhoid  bacilli  into  the  circulation  after 
injuring  the  gall-bladder.  Literature  on  the  relation  of  bacteria  to  gall-stones 
given  by  Funke,  Proc.  Path.  Soc,  Philadelphia,  1908  (11),  17;  also  see  Rosenow 
who  finds  that  streptococci  are  often  responsible  (Jour.  Infect.  Dis.,  1916  (19). 
527).  Grieg  notes  the  frequent  occurrence  of  gall-stones  in  rabbits  immunized 
with  cholera  vibrios  (India  Jour.  Med.  Res.,  1916  (3),  397).  , 


456      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

the  same  time  the  desquamated  cells  and  clumped  bacteria  offer 
suitable  nuclei  upon  which  the  cholesterol  begins  to  crystallize  out. 
Apparently  after  the  calculi  have  reached  a  certain  size  they  cause 
sufficient  mechanical  injury  to  keep  up  the  cell  degeneration  and  chol- 
esterol formation,  even  after  the  infection  has  subsided.  A  certain 
amount  of  infection  and  inflammation  is  a  favoring  condition,  however, 
for  Harley  and  Barratt^*'  found  that  fragments  of  cholesterol  calculi 
introduced  aseptically  into  the  gall-bladders  of  dogs  were  slowly  dis- 
solved and  disappeared,  but  this  was  prevented  by  infecting  the  gall- 
bladder with  B.  coll.  According  to  Naunyn's  investigations,  it  is 
not  an  alteration  in  the  composition  of  the  bile,  as  formed  in  the  liver, 
which  causes  the  precipitation  of  cholesterol,  but  rather  the  presence 
of  the  nidus,  and  the  production  of  large  quantities  of  cholesterol  in 
immediate  proximity  to  this  nidus,  that  determines  the  formation  of  a 
concretion.  In  case  the  bile  stagnates  in  the  gall-bladder,  the  choles- 
terol that  is  being  constantlj^  formed  by  the  normal  disintegration  of 
surface  epithelium  accumulates,  until,  even  without  infection,  there 
forms  a  sediment  of  soft  yellowish  and  brownish  masses,  consisting 
chiely  of  cholesterol  and  bilirubin-calcium.  From  this  material 
calculi  may  eventually  form,  and  by  their  irritation  lead  to  further 
formation  of  cholesterol  and  increased  growth."  But  bacteriological 
studies  indicate  that  generally  an  infectious  influence  is  present  in 
cholelithiasis,  and  bacilli  may  be  found  alive  in  gall-stones  for  remark- 
ably long  periods. 

Recent  applications  of  colloidal  chemistry  add  much  to  our  under- 
standing of  gall-stone  formation.  Thus,  Lichtwitz  points  out  that 
the  colloids  of  normal  bile,  all  of  which  are  electro-negative,  may  be 
precipitated  by  positive  serum  colloids  coming  from  the  blood  when 
the  gall-bladder  is  inflamed;  hence  we  get  a  precipitate  of  cholesterol, 
bilirubin  and  proteins.  When  the  colloids  are  thus  thrown  down  the 
solvent  power  of  the  bile  for  the  alkali  earths  it  contains  is  decreased, 
and  so  calcium  or  magnesium  are  added  to  the  mixture.  Cholesterol 
is  in  solution  in  the  bile  as  an  emulsion  colloid,  and  when  stagnation 
of  the  bile  leads  to  absorption  or  disintegration  of  the  chelates  and 
fats  which  keep  it  in  solution,  the  droplets  become  confluent,  and 
then  crystallization  takes  place  (Schade)  with  formation  of  spheroliths, 
and  eventually  a  crystalline  cholesterol  calculus.  If  even  the  slightest 
pressure  is  brought  to  bear  on  the  myelin-like  masses  before  they 
crystaHize,  however,  they  will  be  pressed  into  scales,  and  the  common 
laminated  structure  results;  hence  crystalline  calculi  are  single,  while 
multiple  gall-stones  are  laminated,  with  perhaps  partial  crj^stallization 
between  the  lamellse.  Also  when  the  gall-stones  result  from  inflamma- 
tion, and  there  is  much  serum  colloid  present,  the  stones  are  lamellated 

88  Jour,  of  Physiol.,  1903  (29),  341;  see  also  Ilansemann,  Virch.  Arch.,  1913 
(212),  139. 

"  Concerning  the  structure  of  gall-stones  see  Ribbert,  Virchow's'  Arch.,  1915 
(220),  20. 


BILIARY  CALCULI  457 

because  these  colloids  deposit  in  that  form  {e.  g.,  corpora  amylacea 
and  other  protein  concretions).  These  considerations  explain  the  for- 
mation of  gall-stones  in  the  gall-bladder  from  either  inflammation, 
or  stagnation  without  inflammation. 

Aschoff  and  Bacmeister,****  however,  hold  that  the  usual  series  of 
events  in  the  formation  of  gall-stones  is  first  the  formation  of  a  pure 
cholesterol  stone  without  inflammatory  cause,  because  of  actual  in- 
creased excretion  of  cholesterol  by  the  liver,  because  of  cholesterolemia; 
or  because  of  resorption  of  solvent  substances  from  stagnating  bile: 
these  primary  cholesterol  stones  then  cause  inflammation  and  occlu- 
sion, leading  to  the  formation  of  the  common  mixed  stones.  Bac- 
meister  ascribes  more  importance  to  calcium  than  do  most  o<"her 
investigators,  in  which  he  is  supported  by  Rosenbloom,*^  while  Kuru^" 
states  that  fibrin  is  usually  present.  Boscnbloom  reports  a  small 
series  in  which  concretions  composed  chiefly  of  calcium  were  found  in 
all  cases  with  a  history  of  infection,  while  in  cases  without  infection 
the  stones  we;*e  cholesterol. 

More  recent  studies  of  the  cholesterol  content  of  the  blood  and  bile 
also  have  reacted  against  the  concept  that  all  the  cholesterol  of  gall- 
stones comes  from  the  wall  of  the  bile  tract  through  inflammatory 
changes.  It  has  been  found  that  patients  with  gall-stones  often  show 
a  hypercholesterolemia;^^  that  pregnancy,  which  seems  to  be  a  predis- 
posing cause  of  cholelithiasis,  is  accompanied  by  hypercholesterolemia; 
that  in  races  subject  to  cholelithiasis  there  is  more  cholesterol  in  the 
diet  and  in  the  blood  than  in  those  races  that  seldom  have  gall-stones 
(DeLangen) ;  that  with  hypercholesterolemia  there  is  an  increased  out- 
put of  cholesterol  in  the  bile,  and  that  experimental  hypercholester- 
olemia may  lead  to  the  formation  of  gall-stones  without  evident 
infection  of  the  bile  tracts  (Dewey^^). 

As  far  as  the  existing  evidence  permits  one  to  draw  conclusions,  it 
would  seem  probable  that  both  local  and  systemic  conditions  are  of 
iniyortance  iii  gall-stone  formation.  Apparently,  gall-stones  may  form 
from  cholesterol  derived  from  the  inflamed  bile  tract  walls,  independent 
of  the  amount  of  cholesterol  present  in  the  bile;  but  presumably  they 
may  derive  part  if  not  all  the  cholesterol  from  the  bile  in  some  cases. 
In  either  event,  a  hj^percholesterolemia  will  favor  their  formation, 
and  hence  any  given  condition  of  injury  to  the  gall  bladder  will  more 
often  give  rise  to  concretions  in  persons  with  a  high  cholesterol  content 
in  the  blood.  ^^     Changes  in  the  bile  itself  may  be  produced  by  disease 

88  Ziegler's  Beitr.,  1908  (44),  528. 

89  Jour.  Amer.  Med.  Assoc,  1917  (69),  1765. 
»"  Virchow's  Arch.,  1912  (210),  433. 

»i  Henes,  Surji.,  Gyn.  and  Obst.,  1916  (23),  91. 

»2  Arch.  Int.  Med.,  1916  (17),  757;  see  also  Aoyama,  Deut.  Zeit.  Chir.,  1914 
(132),  234. 

9^  This  relation  of  hypercholesterolemia  and  infection  to  cholelithiasis  is  sup- 
ported by  the  extensive  observations  of  Rothschild  and  Wilensky  (Anier.  Jour. 
Med.  Sci.,  1918  (156),  239,  404,  564;  Arch.  Int.  Med.,  1919  (24),  520),  who  find 
some  types  of  cases  accompanied  by  cholesterol  increase,  which  is  missing  in 
many  cases  of  inflammatory  cholelithiasis.  (See  also  Reimann  and  Magown, 
Surg.,  Gynec.  and  Obst.,  1918  (26),  282;  Fasiani,  Arch.  Sci.  Med.,  1918  (41),  144. 


458      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

of  the  liver  that  will  alter  bile  compositon  in  such  a  way  that  its  capac- 
ity to  sustain  cholesterol  in  solution  or  suspension  will  be  lowered, ^"^ 
and  this  factor  also  cannot  be  dismissed  as  without  importance;  tran- 
sient thickening  of  the  bile,  such  as  may  occur  in  any  febrile  disease, 
may  also  very  possibly  initiate  precipitation  and  stone  formation. 
More  and  more  this  last  factor  is  receiving  consideration,  together  with 
hypercholesterolemia,  as  of  importance  in  producing  cholelithiasis. 
Rovsing,^^  quoting  Boysen's  analysis  of  200  autopsy  cases  of  choleli- 
thiasis, which  showed  that  all  recent  deposits  and  the  centers  of  older 
concretions  consisted  of  calcium-pigment,  especially  emphasizes  this 
transitory  concentration  of  bile. 

It  was  formerly  supposed  that  the  calcium-pigment  concretions 
were  produced  by  the  presence  of  excessive  calcium  in  the  bile,  derived 
particularly  from  lime-laden  drinking-water,  but  it  has  been  demon- 
strated that  increase  of  calcium  in  the  food  does  not  cause  an  increase 
in  the  amount  in  the  bile.  Furthermore,  on  concentrating  bile, 
which  contains  both  bilirubin  and  calcium,  the  free  bilirubin  separates 
out  and  not  the  calcium  compound  of  bihrubin;  and  also,  Naunyn 
found  that  the  bile  salts  prevent  precipitation  of  calcium-bilirubin, 
even  when  calcium  salts  are  added  in  considerable  amounts.  Appar- 
ently it  is  the  presence  of  positively  charged  protein  substances  that 
leads  to  the  precipitation  of  this  electro-negative  substance  from  bile, 
and  hence  the  formation  of  pigment  calculi  is  also  favored  or  initiated 
by  inflammation  of  the  bile  tracts,  particularly  as  most  of  the  calcium 
salts  seem  to  come  from  the  mucous  membrane ;^'^  later,  as  we  have  seen, 
these  pigment  concretions  often  become  covered  with  cholesterol 
derived  from  the  injured  epithelium,  and  the  common  mixed  calculi 
are  then  formed.  In  view  of  the  fact  that  much  of  the  pigment  in  these 
calculi  is  composed  of  the  oxidation  products  of  bilirubin,  especially 
bilihumin,  it  is  possible  that  oxidation  processes  in  the  stagnating 
bile  are  important  causes  of  the  precipitation;  Naunyn  suggests  that 
bacteria  may  be  the  cause  of  the  oxidation.  Pigment  calculi  are  par- 
ticularly important  as  the  starting-point  of  the  larger  mixed  calculi. 
Aufrecht,^^  indeed,  holds  that  gall-stone  formation  usually  begins  with 
particles  of  pigment  that  are  expelled  from  the  liver  cells  as  such, 
and  ordinarily  are  discharged  into  the  intestine;  if  they  make  their 
way  back  into  the  gall-bladder  they  form  the  nuclei  of  concretions. 
It  is  possible,  Naunyn  believes,  for  the  pigment  to  be  later  gradually 
replaced  by  cholesterol. 

"^  See  D'Amato,  Biochcm.  Zoit.,  191.'5  (G9),  353. 

'•^  Hospitalstidende,  1915  (5S),  249. 

""  This  commonly-held  view  is  denied  by  Liehtwitz  and  Book  (Dent.  med. 
Woch.,  1915  (41),  1215),  who  fonnd  the  calcium  content  of  bile  from  fistnlns  to 
be  from  65-84  mg.  per  liter,  and  in  bladder  l)ile  to  vary  from  85  to  325  mg.,  but 
not  according  to  the  presence  or  absence  of  inflammation. 

»'  Deut.  Arch.  klin.  Med.,  1919  (128),  242. 


URINARY  CONCRETIONS  459 

Urinary   Calculi'* 

These  differ  from  the  bile  concretions  in  two  important  respects: 
First,  there  is  no  evidence  that  any  considerable  part  of  their  con- 
stituents may  come  from  the  walls  of  the  cavities  that  contain  them ; 
they  are  usuall}^  deposited  on  account  of  an  over-saturation  of  the 
urine,  or  on  account  of  a  change  in  composition  of  the  urine,  which 
renders  them  insoluble.  Second,  the  composition  of  urinary  calculi  is 
usually  less  mixed  than  that  of  biliary  calculi,  although  seldom,  if  ever, 
is  it  pure.  Thus,  Finstcrer  found  but  six  concretions  composed  of 
onlj'  one  substance,  in  a  collection  of  114  calculi.  As  with  the  bile,  the 
chief  constituent  of  the  urine  (urea)  is  so  soluble  that  it  never  forms 
concretions,  but  only  the  less  soluble  minor  constituents  are  thrown 
down.  For  the  formation  of  calculi,  however,  it  is  not  sufficient  to 
have  merely  an  excess  of  a  substance  in  the  urine,  for  we  may  have 
deposition  of  urates,  phosphates,  or  uric  acid  in  simple  crystalline 
form  without  the  formation  of  calculi.  A  nucleus  of  some  sort  is 
present  as  well  as  a  binding  suhsiance,^^  which  is  often  mucus  derived 
from  the  walls  of  the  passages,  although  the  center  of  the  concretion 
most  often  consists  of  uric  acid  or  urates. 

Although  the  amount  of  colloidal  material  in  urine  is  relatively 
small,  yet  it  undoubtedly  plays  an  important  part  in  maintaining  in 
solution  the  less  soluble  crystalloids,  which  are  especially  the  urates  and 
calcium  oxalate.  Normal  urine  contains  no  colloids  which  form  irre- 
versible gels,  and  hence  ordinary  deposits  can  be  readily  dissolved,  but 
in  inflammatory  conditions  there  appears  fibrinogen  which  readily 
forms  the  irreversible  fibrin,  and  conditions  thus  become  favorable  for 
the  formation  of  concretions  of  any  crystalloid  with  which  the  urine 
may  be  saturated  or  over-saturated  at  the  time  (Schade).  Possibly 
other  colloids  may  play  a  similar  role.  Aschoff  and  Kleinschmidt' 
hold  that  most  urinary  calculi  begin  as  primary  calculi,  formed  inde- 
pendent of  inflammation  from  excess  of  the  main  constituent  (uric  acid, 
oxalates,  xanthine,  but  chiefly  ammonium  urate) ;  this  calculus  forms 
the  crystalline  nucleus  of  the  laminated  secondary  deposits  of  other 
substances,  chiefly  uric,  acid,  oxalates  and  phosphates,  all  being 
deposited  without  inflammation.  The  inflammatory  formations  con- 
sist chieflj'^  of  ammonio-magnesium  phosphate  and  ammonium  urate, 
usually  deposited  on  a  foreign  body  or  a  primarj^  calculus.  The  ex- 
tensive study  of  the  microscopic  structure  of  urinary  calculi  by 
Shattock,-  shows  also  that  a  nucleus  of  cells  or  other  organic  material 
is,  at  least  in  uric  acid  calculi,  extremely  rare,  the  center  being  almost 
always  a  primary  crystalline  deposit  from  a  supersaturated  solution. 

"*  General  Bibliography  given  by  Finsterer,  Deut.  Zeit.  klin.  Chir.,  1906  (80), 
41 -4;  and  Lichtwitz.'= 

^'  Hippocrates  appreciated  the  existence  and  importance  of  the  mucoid  binding 
substance  in  urinary  concretions  (Schepelmann,  Berl.  klin.  Woch.,  1911  (48),  525). 
^"Die  Harnsteine,"  Berlin,  Julius  Springer,  1911. 

2Proc.  Roy.  Soc.  Med.,  Path.  Sec,  1911  (4),  110. 


460      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

Calculi  formed  because  of  changes  in  the  urinary  composition  in- 
dependent of  evident  infection  are  often  called  "primary,"  in  con- 
tradistinction to  those  arising  from  changes  in  composition  brought 
about  by  infection  and  ammoniacal  decomposition.  Because  of  the 
injury  produced  by  a  primary  calculus,  infection  frequently  results, 
and  then  the  primary  calculus  may  become  the  nucleus  of  a  secondary 
calculus;  indeed,  on  account  of  the  change  of  reaction,  the  crystalloids 
of  the  primary  calculus  may  be  dissolved  out,  and  their  place  taken  by 
the  secondary  deposit  {metamor'phosed  calculi).  In  structure  urin- 
ary calculi  usually  show  both  radiating  and  concentric  lines  of  forma- 
tion, and  when  the  chief  constituents  are  dissolved  away,  an  organic 
framework  remains.  They  are  generally  classified  according  to  their 
prominent  component,  as  follows: 

Uric=Acid  Calculi. — Uric  acid  is  but  shghtly  soluble,  only  one 
part  dissolving  in  39,480  of  pure  water  at  18°,  and  it  is  even  less  soluble 
in  the  presence  of  acids.^  The  presence  of  sodium  diphosphate  in  the 
solution  makes  it  much  more  soluble,  and  various  organic  bodies  also 
favor  its  solution,  among  them  being  the  urinarj^  pigments.  As  can 
be  seen,  the  maintenance  of  uric  acid  in  solution  is  by  a  small  margin, 
even  in  normal  conditions;  hence  the  mere  cooling  of  the  urine  fre- 
quently suffices  to  cause  an  abundant  deposition  of  uric  acid  combined 
with  pigment,  as  the  familiar  "brick-dust"  deposit.  The  formation 
of  uric-acid  calculi  is,  therefore,  not  only  a  question  of  the  amount  of 
uric  acid  in  the  urine,  but  depends  even  more  upon  the  amount  of  the 
substances  that  hold  it  in  solution,  and  as  both  these  factors  are  sub- 
ject to  wide  variations  under  both  physiological  and  pathological  con- 
ditions, uric  acid  and  urates  are  common  in  urinary  concretions. 

The  older  literature  indicates  that  the  most  common  calculus  is  of 
this  nature,  but  a  number  of  recent  analyses  indicate  that  the  im- 
portance of  uric  acid  and  urates  has  been  overestimated.  On  the  con- 
trary, this  material  rarely  forms  a  considerable  part  of  the  calculi, 
but  is  usually  present  in  greater  or  less  amount  in  most  or  all  urinary 
calculi  (Kahn).^  It  is  probable,  however,  that  uric  acid  is  important 
as  furnishing  the  primary  nucleus  of  calculi  of  preponderatingly  cal- 
careous or  mixed  composition.  Apparently  there  are  marked  differ- 
ences in  the  prevailing  composition  of  calculi  in  different  countries;  in 
China,  for  example,  Pfister'^  found  eleven  of  twelve  calculi  composed 
of  uric  acid. 

Uric  acid  is  eliminated  combined  chiefly  with  sodium,  potassium, 
and  ammonium;  according  to  some  authors,  as  abiurate,  according  to 
others,  as  a  quadriurate.     If  the  urine  is  excessively  acid,  it  con- 

^  Concerning  solubility  of  uric  acid  in  urine  see  Haskins,  Jour.  Biol.  Chem., 
1916  (26),  205. 

*  Arch.  Int.  Med.,  '1913  (11),  92;  review  of  literature.  Rosenbloom,  (Jour. 
Amer.  Med.  Assoc,  1915  (65),  161)  found  but  two  uric  acid  stones  of  twenty-six 
analyzed. 

6  Zeit.  Urol.,  1913  (7),  915. 


URINARY  CONCRETIONS  461 

tains  much  acid  j^hospliates,  wliicli  withdraw  part  of  the  bases  from 
the  uric  acid,  and  this,  when  free,  crystalhzcs  out  if  in  excess.  Hence 
the  formation  of  uric-acid  concretions  is  favored  by  high  acidity  of 
the  urine,  by  concentration  of  the  urine,  or  by  an  increased  ehmina- 
tion  of  the  uric  acid.  The  last  may  result  from  excessive  nuclein- 
rich  food,  or  from  excessive  catabolism  of  the  tissue  nucleoproteins 
(e.  g.,  leucocytosis  from  inflammatory  diseases  or  leukemia),  which 
conditions  are  also  usually  associated  with  an  increased  urinary  acid- 
ity. The  chemistry  of  uric  acid  is  discussed  more  fully  in  the  chap- 
ter on  Gout,  Chap,  xxiii.) 

Uric-acid  calculi  are  formed  chiefly  in  the  pelvis  of  the  kidney,  but 
many  pass  into  the  bladder.  They  are  quite  hard,  and  yellow  or 
reddish-yellow  in  color,  because  of  the  presence  of  urochrome  and 
urobilin,  the  former  of  which  seems  to  be  chemically  combined  and 
the  latter  but  physically,  since  it  can  be  washed  out  with  water. 
Uroerythrin  or  uromelanin  (a  decomposition  product  of  urochrome) 
may  also  be  present.  Not  infrequently  calcium  oxalate  is  present, 
.sometimes  in  considerable  quantities.  Other  urinary  constituents  may 
be  present  in  small  amounts.  In  case  the  calculus  enters  the  urinary 
bladder  it  may  set  up  irritation  leading  to  infection;  the  urine  then 
becoming  alkaline,  calcium  and  ammonio-magnesium  phosphate  will 
be  deposited  upon  the  surface,  and  the  uric  acid  will  be  more  or 
less  dissolved  out  and  replaced  by  the  phosphates  (metamorphosis). 

Urate  calculi  occur  chiefly  in  new-born  or  young  infants,  and 
rarely  in  adults.  In  the  young  they  are  related  to,  and  may  originate 
in,  the  deposits  of  urates  in  the  pyramids  of  the  kidney  (the  so-called 
urate  or  uric-acid  "infarcts"),  which  have  been  supposed  to  result 
from  the  decomposition  of  the  nucleoproteins  of  the  nucleated  fetal 
red  corpuscles.  (See  Uric  Acid,  Chap,  xxiii.)  The  concretions  are 
composed  chiefly  of  either  ammonium  or  sodium  urate,  but  potassium 
and  even  calcium  and  magnesium  urate  may  be  admixed.  Their 
genesis  in  the  young  probably  depends  upon  injury  to  epithelium  by 
the  excessive  urates  of  the  "infarcts,"  which  affords  a  suitable  nucleus 
for  their  start;  their  growth  depends  chiefly  upon  the  concentration  of 
the  infant's  urine.  In  adults  they  may  arise  secondary  to  an  am- 
moniacal  decomposition  of  the  urine.  Urate  concretions  are  not  com- 
mon; they  are  generally  rather  soft,  and  often  much  colored  b}' 
pigments. 

Calcium  oxalate  calculi  are,  according  to  recent  observers,*  the 
most  common  urinary  concretions.^  Often  they  show  admixtures  of 
urates  or  uric  acid,  which  latter  frequently  constitutes  the  nucleus,  and 
when  urinary  infection  occurs  they  may  in  turn  serve  as  the  nucleus 
to  phosphatic  deposits.  On  account  of  the  hardness  and  roughness  of 
tliese  stones  they  frequently  cause  bleeding,  which  may  result  in  their 

^  Concerning  their  structure  see  Fowler,  Johns  Hopkins  Hospital  Reports,  1908 
(13),  507. 


462     CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

being  very  dark  in  color  and  containing  blood-pigment.  They  are 
usually  first  formed  in  the  pelvis  of  the  kidney,  and  arise  chiefly  in 
persons  excreting  excessive  quantities  of  oxalic  acid.  Normally  but 
about  0.02-0.05  gram  of  oxalic  acid  is  eliminated  daily  in  the  urine, 
apparently  all  as  calcium  oxalate,  which  is  kept  in  solution  by  the 
acid  phosphates.  The  amount  may  be  increased  by  certain  foods 
rich  in  oxalates,  particularly  rhubarb,  grapes,  spinach,  etc.;  also  prob- 
ably by  gastric  fermentation.'^  Oxalic  acid  may  possibly  be  formed 
from  uric  acid,  and  perhaps  also  from  the  carbohydrate  group  of 
proteins,^  and  it  is  possible  that  abnormally  large  amounts  arise  from 
these  sources  under  pathological  conditions.  During  bacterial  de- 
composition of  the  urine  oxalic  acid  may  be  formed  from  uric  acid 
(Austin).  9 

Phosphate  calculi  are  formed  as  a  result  of  decomposition  of  the 
urine,  with  formation  of  ammonia  from  the  urea.^"  In  the  ammoniacal 
solution  thus  formed  the  magnesium  is  precipitated  as  NH4MgP04, 
the  calcium  as  Ca3(P04)2,  and  calcium  oxalate  and  ammonium  urate 
are  also  thrown  down,  so  that  the  concretions  consist  of  a  mixture  of 
these  substances,  the  magnesium  salt  being  the  most  abundant.  In 
none  does  one  substance  occur  in  a  pure  state.  Pigments  of  various 
kinds,  and  more  or  less  mucus  or  other  organic  constituents  of  the 
framework  are  also  present.  Phosphate  calculi  are  the  typical  "sec- 
ondary" concretions,  and  they  are  formed  usually  in  the  bladder  as  a 
consequence  of  cystitis,  but  may  be  formed  in  the  renal  pelvis  or  in 
the  urethra.  In  some  cases  the  salts  are  precipitated  in  such  large 
quantities  that  they  form  great  masses  of  a  sediment  which  does  not 
aggregate  into  concretions.  Occasionally  stones  consisting  princi- 
pally of  Ca3(P04)2  or  CaHP04  are  formed,  but  these  are  rarities.  As 
the  calcium  taken  in  the  food  is  chiefly  eliminated  in  the  feces,  the 
amount  in  the  urine  does  not  vary  directly  with  the  amount  in  the 
food,  and  the  formation  of  phosphatic  concretions  is  always  a  matter 
or  urinary  reaction  and  not  of  diet.^'  .4s  these  stones  fuse  to  a  black, 
enamel-like  mass  under  the  blow-pipe,  they  have  been  called  "fusible 
calculi." 

Calcium  carbonate  calculi  are  formed  frequently  in  herbivora,  but  they  are 
very  rare  in  the  urinary  passages  of  man,  although  occurring  elsewhere  intliebody 

'  Baldwin,  Jour.  Exp.  Med.,  1900  (5),  27. 

*  See  Austin,  Boston  Med.  and  Surg.  Journal,  1901  (145),  ISl.     Contradicted 
by  Wegrzynowski,  Zeit.  physiol.  Chem.,  1913  (83),  112. 
9  Jour.  Med.  Research,  1906  (15),  314. 

">  Under  the  name  "struvit  stone,"  Pommer  (Vcrh.  deut.  Path.  Gesell.,  1905 
(9),  28)  describes  a  urinary  calculus  composed  of  very  pure  ammonio-magnesium 
phosphate,  forming  the  hard,  rhoml)ic  crystals  known  to  mineralogists  as  "struvit." 
This  is  an  example  of  a  phosphate  stone  formed  independent  of  ammoniacal  decom- 
position, a  rare  occurrence. 

•'Osborne  (Jour.  Amer.  Med.  Assoc,  1917  (69),  32)  has  observed  numerous 
cases  of  formation  of  phosphate  calculi  in  tlie  urinary  bladder  of  rats  kept  on  diets 
deficient  in  fat  soluble  vitamines.  The  reason  for  this  association  of  diet  and 
concretions  is  not  known;  possibly  the  dietary  deficiency  causes  lessened  resist- 
ance to  urinary  infection. 


URINARY  CONCRETIONS  463 

not  infrequently.  Occasionally  these  arc  soft  and  chalky,  hut  if  well  crystallized, 
they  are  the  liartlost  of  concretions. 

Cystine  calculi'-  are  rare  but  very  interesting  formations.  Cystine 
S-CH(N1I...)-C()()1I 

I  is  important  as  the  sulphur-containing  portion  of  the  protein 

S-CII(NH2)-C()0II 

molecule.  Under  normal  conditions  all  the  cystine  taken  in  food  is  completely  oxi- 
dized and  none  (or  uncertain  traces)  appears  in  the  urine.  In  certain  individuals 
the  urine  contains  considerable  quantities  of  cystine  constantly  {cyslinuria,  see 
Chap  xxi),  and  occasionally  in  these  cases  soft  concretions  of  nearly  pure  cystine 
are  formed  in  the  urinary  passages.  Cystine  calculi  may  reach  the  size  of  a  hen's 
egg,  are  crystalhne  in  structure,  and  in  the  urine  of  such  patients  the  characteristic 
hexagonal  crystals  may  usually  be  found.  The  cystine  of  calculi  is  identical  with 
that  from  proteins  and  may  be  associated  with  tyrosine.'^ 

Xanthine  Calculi — Xanthine  is  the  most  abundant  of  the  purine  bases  normally 
present  in  urine,  but  the  total  amount  is  extremely  small.  Like  uric  acid,  it  fluc- 
tuates in  amount  according  to  the  amount  of  destruction  of  nucleoproteins,  either 
of  the  food  or  of  the  tissues.  Concretions  consisting  chiefly  of  xanthine,  which  is 
often  mixed  with  uric  acid,  are  extremely  rare,  but  a  few  isolated  specimens  having 
been  described.  Rosenbloom  could  collect  but  six  cases  in  the  literature,  adding 
one  himself.'* 

Indigo  calculi,  derived  from  the  indican  of  the  urine  through  oxidation,  have 
also  been  described  a  few  times. 

Urostealith  calculi,  composed  of  fatty  matter,  have  been  occasionally  observed. 
Although  some  of  the  concretions  described  under  this  head  have  really  repre- 
sented foreign  bodies  introduced  through  the  urethra  (e.  g.,  Kruckenberg's  concre- 
tion of  paraffin  from  a  bougie),  yet  true  fat  concretions  do  occur.  The  origin  of  the 
fat  in  these  stealiths  is  unknown;  possibly  it  comes  from  degenerated  epithelium. 
Horbaczewski'5  analyzed  such  a  specimen  which  had  the  following  percentage 
composition: 

Water 2.5 

Inorganic  matter 0.8 

Organic  matter  (chiefly  protein) 11.7 

Fatty  acids 51.5 

Neutral  fat 33 . 5 

Cholesterol traces 

The  fatty  acids  consisted  of  stearic,  palmitic,  and  probably  myristic  acid. 

Cholesterol  calculi  have  been  found  in  the  urinary  bladder  in  a  few  instances, 
the  cause  being  unknown.  Horbaczewski^*  describes  one  weighing  25.4  grams, 
found  in  a  patient  who  had  previously  had  cystine  calculi;  it  contained  95.87  per 
cent,  of  cholesterol  and  but  0.55  per  cent,  of  inorganic  material.  Gall-stones  have 
been  known  to  enter  the  urinary  bladder  through  a  fistula  between  the  gall-bladder 
and  urinary  bladder.  ^^ 

Fibrin  "calculi,"  formed  from  blood-clots,  often  more  or  less  impregnated  with 
urinary  salts,  have  occasionally  been  observed.  Other  proteins  may  also  form  simi- 
lar calculi.'" 

General  Properties  of  Urinary  Concretions.'^ — The  hardness 
depends  partly  upon  the  chemical  composition  of  the  calculus,  but 
more  upon  the  rate  and  condition  of  formation  (Rowlands,  Kahn). 

1-  Literature  concerning  cystine,  see  Friedmann,  Ergeb.  der  Physiol.,  1902  (i), 
15;  Marriott  and  Wolf,  Am.  Jour.  Med.  Sci.,  1906  (131),  197. 

13  Abderhalden,  Zeit.  physiol.  Chem.,  1907  (51),  391;  1919  (10-1),  129. 

»  N.  Y.  Med.  Jour.,  Jan.  16,  1915. 

's  Zeit.  physiol.  Chem.,  1894  (18),  335. 

16  See  Finsterer,  Deut.  Zeit.  klin.  Chir.,  1906  (80),  426. 

1^  See  Morawitz  and  Adrian,  Mitt.  Grenz.  Med.  u.  Chir.,  1907  (17),  579. 

1*  Systems  for  procedure  in  determining  the  nature  of  urinary  calculi  are  given 
by  Hammarsten  (Text-book  of  Phj'siol.  Chem.)  and  by  Smith  (Reference  Hand-book 
of  Med.  Sci.). 


464      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

Under  comparable  conditions  it  is  said  that  those  composed  of  amorph- 
ous phosphates  are  the  softest;  next  come  those  with  some  admixture 
of  crystalhne  phosphates.  Urate  concretions  are  harder  than  these, 
but  are  still  softer  than  uric  acid  and  crystalline  phosphate  calculi. 
Oxalates  are  usually  the  hardest,  except  for  the  rare  crystallized 
calcium  carbonate  stones.  Cystine  and  amorphous  concretions  can 
be  scratched  with  the  finger-nail,  while  even  the  hardest  varieties  of 
calculi  can  be  scratched  with  a  wire  nail.  Genersich^^  gives  the 
following  degrees  of  hardness  for  different  calculi:  Cholesterol,  1,5- 
1.6;  ammonium  urate,  2.5;  soft  phosphate  (Mg),  2.6;  hard  phos- 
phate (Ca),  2.75;  uric-acid  stones  (also  salivary  and  prostatic  calculi, 
atheromatous  patches,  and  phleboliths),  2.9;  calcium  oxalate  (also 
rhinoliths  and  lung  stones),  3.3-3.5;  calcium  carbonate  stones  of 
herbivora,  4.5.  But  the  hardness  or  gross  appearance  of  a  urinary 
calculus  give  little  or  no  indication  of  its  chemical  composition. 

The  rate  of  growth  also  varies  according  to  composition,  but  is,  of 
course,  much  modified  by  other  factors.  Oxalate  and  urate  stones 
grow  most  slowly,  phosphate  stones  most  rapidly.  A  urate  stone  has 
been  known  to  increase  by  about  two  ounces  during  seven  and  one 
half  years,  while  a  catheter  fragment  or  other  foreign  body  may  be- 
come covered  with  a  crust  several  millimeters  thick  in  a  few  weeks. ^"^ 

Spontaneous  disintegration  of  urinary  concretions  is  limited  almost  solely  to 
calculi  composed  entirely  or  largely  of  uric  acid.  Out  of  121  cases  collected  by 
Englisch.^i  in  all  but  7  this  was  the  case,  these  being  composed  of  calcium  and 
magnesium  phosphate  (5),  or  calcium  phosphate  or  carbonate  (1  each).  The 
disintegration  is  brought  about  through  solution  of  the  binding  substance  and  me- 
chanical shattering  of  the  stone  into  fragments.  This  occurs  but  rarely,  Bastos- 
estimating  that  perhaps  one  calculus  in  ten  thousand  undergoes  disintegration. 

Corpora  Amylacea^^ 

In  the  case  of  these  widely-spread  concentric  bodies  we  find  the 
name  misleading,  for  the  bodies  are  not  a  form  of  animal  starch,  as 
was  suggested  by  their  laminated  structure  and  iodiu  reaction,  nor 
are  they  so  closely  related  to  amyloid  material  as  the  name  implies. 
Different  authors  disagree  decidedly  concerning  the  staining  reactions 
of  these  bodies,  but  it  may  be  said  that  the  reactions  are  extremely 
inconstant.  Sometimes  the  corpora  are  stained  bluish  or  green  with 
iodin,  sometimes  brown,  often  little  at  all;  occasionally  they  react 
partly  with  methyl- violet,  but  more  often  they  do  not;  sometimes  por- 
tions of  one  body  react  one  way,  while  the  remainder  behaves  differ- 
ently.    Seldom  if  ever  do  the  ordinary  concretions  of  the  prostate 

"  Virchow's  Arch.,  1893  (131),  185. 

20  ZuckerkandLNothnagel's  System,  vol.  19,  pt.  2,  p.  229. 

21  Arch.  klin.  Chir.,  1905  (76),  961  (elaborate  revie^v). 
"  Folia  Urol.,  1913  (8),  81. 

"  General  literature,  Posner,  Zeit.  klin.  Med.,  1889  (16),  144;  Lubarsch, 
Ergeb.  allg.  Pathol.,  1894  (lo)  180;  Ophiils,  Jour.  Exp.  Med.,  1900  (5;,  111; 
Nunokawa,  Virchow's  Arch.,  1909  (196),  221;  Brutt,  ibid.,  1912  (207),  412. 


CORPORA   A.^rVLACEA  465 

give  all  the  amyloid  reactions  characteristically,  but  the  corpora 
amylacea  of  the  lungs  are  much  more  likely  to  do  so  (Stumpf).^'  It 
seems  improbable  that  these  bodies,  which  occur  in  the  prostate  of 
every  adult,  can  be  the  same  as  the  amyloid,  which  is  seldom  observed 
except  as  the  result  of  serious  processes  of  tissue  destruction.  Accord- 
ing to  their  structure  they  obey  the  usual  laws  of  the  formation  of 
concretions,  having  a  central  nucleus  and  a  structural  framework  of 
different  composition  from  the  chief  substance.  It  seems  most  prob- 
able that  they  should  bo  interpreted  as  simple  concretions  of  protein 
nature,  which  form  under  certain  conditions  when  a  nucleus  of  some 
sort  (usually  pigment,  degenerated  cells,  or  inorganic  crystals)  exists 
in  a  stagnating,  protein-rich  fluid.  At  limcs  the  resulting  concretion 
may  be  of  such  a  physical  nature  that  it  absorbs  iodin  readil}^  (just 
as  they  often  show  a  marked  absorption-affinity  for  pigments),  and 
occasionally  it  may  react  metachromatically  with  methyl-violet,  pos- 
sibly because  of  the  presence  of  chondroitin-sulphuric  acid  derived 
from  the  mucin  of  the  cavities  where  the  concretions  form,  but  per- 
haps for  some  other  unknown  reasons.  Occasionally  pure  amyloid 
may  form  in  the  tissues  typically  concentric  (or  even  crystalline) 
bodies,  as  in  Ophiil's  case,  but  this  is  the  exception.  It  seems  prob- 
able that  corpora  amylacea  are  usually  protein  concretions,^'^  and 
neither  amyloid  nor  animal  starch.  Those  formed  in  the  central 
nervous  system  may  be  of  myelin  or  neuroglia  origin.-^ 

The  small  amount  of  material  available  prevents  an  accurate 
analysis  of  the  corpora  amylacea;  it  is  known  that  they  are  very  in- 
soluble in  water,  acids,  alkalies,  etc.,  behaving  like  coagulated  protein 
in  this  respect.  Even  hot  concentrated  nitric  acid  will  not  dissolve 
them,  according  to  Posner.  This  author  considers  lecithin  and  cho- 
lesterol to  be  important  constituents,  and  by  Ciaccio's  staining 
method  lipoids  can  be  found  in  prostatic  corpora  amylacea. ^^  How- 
ever, it  is  said  by  Bjorling-^  that  the  ordinary  hyaline  and  granular 
corpora  do  not  contain  fats  or  lipoids,  but  that  a  certain  class  of 
"lipoid"  prostatic  concretions  contain  many  granules  of  this  nature. 
The  corpora  amylacea  of  the  lateral  ventricles  seem  to  consist  chiefly 
of  calcium  salts  deposited  in  a  concentric  arrangement  through  the 
medium  of  an  organic  basis.  Posner  considers  that  the  presence  of 
lecithin  in  prostatic  corpora  prevents  their  calcification,  although 
this  change  occasionally  does  occur. 

Other  Less  Common  Concretions 
Pancreatic  Calculi.-^ — The  cause  of  the  formation  of  stones  in  the  pancreatic 
duct  is  not  definitely  known,  but  apparently  infection  is  the  most  important  factor, 

"  Virchow's  Arch.,  1910  (202),  134. 

"  Ramsden's  observations  (Proc.  Royal  Soc,  1903  (72),  156)  on  the  precipi- 
tation of  proteins  by  the  action  of  surface  contact  may  have  some  bearing  on 
the  formation  of  such  protein  concretions. 

2«  See  Lafora,  Virchow's  Arch.,  1911  (205),  295. 

"  Posner,  Zeit.  f.  Urologie,  1911  (5),  722. 

28 /bid,  1912  (6)    30. 

2^  Literature  by  Scheunert  and  Bergholz,  Zeit.  phj'siol.  Chem.,  1907  (52),  338. 

30 


466      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 

since  simple  experimental  stasis  will  not  cause  their  formation.'"  The  calculi 
consist  usually  of  a  mixture  of  calcium  phosphate  and  carbonate,  associated  with 
more  or  less  organic  matter,  including  frequently  cholesterol,  but  all  the  usual 
products  of  proteolysis  may  be  present  because  of  the  presence  of  trypsin.  Oc- 
casionally the  calculi  consist  chiefly  of  calcium  carbonate,  which  may  be  almost 
pure.'^  Shattock^2  ^^g  observed  a  pancreatic  concretion  composed  of  calcium 
oxalate.  Sodium  phosphate  and  chloride,  magnesium  phosphate,  and  proteins 
have  also  been  found  in  these  concretions.  Taylor^'  describes  a  pancreatic  con- 
cretion containing,  according  to  the  analyst,  chiefly  silicate  (!),  a  finding  difficult  to 
understand  or  accept. 

Baldoni'^  found,  on  analysis  of  a  stone  weighing  3.1  grams,  the  following 
percentage  composition: 

Water 3 .  44 

Ash 12.67 

Proteins 3 .  49 

Free  fatty  acids 13 .  39 

Neutral  fatty  acids 12 .  40 

Cholesterol 7 .  69 

Pigments  and  soap 40 .  91 

Undetermined 6.01 

Usually,  however,  pancreas  stones  consist  chiefly  of  inorganic  substances. 
Johnson  and  WoUaston  report  analyses  of  two  stones,  one  containing  72.30  per 
cent,  calcium  phosphate  and  but  8.80  per  cent,  organic  matter;  the  other  91.65 
per  cent,  calcium  carbonate,  4.15  per  cent,  magnesium  carbonate,  and  but  3  per 
cent,  organic  matter.  Legrand'^  found  only  0.7  per  cent,  organic  matter  in  another 
concretion  which  contained  93.1  per  cent,  calcium  carbonate.  Pancreatic  juice 
being  strongly  alkaline,  can  hold  but  a  small  quantity  of  calcium  salts  in  solution 
(normally  but  0.22  part  per  thousand — C.  Schmidt);  presumably  the  little  nor- 
mally present  is  held  in  the  form  of  a  colloidal  suspension  by  the  proteins.  Possi- 
bly when  stasis  occurs,  digestion  of  the  proteins  leads  to  the  precipitation  of  the 
calcium  salts,  or,  more  probably,  the  excessive  calcium  is  largely  derived  from  the 
exudate  from  the  inflamed  ducts,  as  seems  to  be  the  case  with  the  calcium  of  biUary 
calculi. 

Salivary  Calculi.'^ — These  have  a  similar  composition,  in  the  main,  to  the  con- 
cretions of  the  pancreatic  duct,  except  that  they  generally  contain  more  organic 
matter,  resembling  in  this  respect  the  "tartar"  of  the  teeth."  Bessanez  found 
in  one  81.3  per  cent,  of  calcium  carbonate  and  4.1  per  cent,  of  calcium  phosphate, 
whereas  in  another  the  carbonate  was  but  2  per  cent,  and  the  phosphate  75  per 
cent.  Potties  has  described  a  calculus  with  a  central  portion  composed  chiefly 
of  uric  acid  and  a  peripheral  portion  containing  69  per  cent,  of  calcium  phosphate 
and  20.1  per  cent,  of  calcium  carbonate.  Harlay^*  found  in  one  specimen  15.9 
per  cent,  organic  matter,  75.3  per  cent,  calcium  phosphate,  6.1  per  cent,  calcium 
carbonate.  Roberg  believes  that  bacteria  alone  do  not  usually  cause  salivary 
calculi  to  form,  but  that  a  foreign  body  entering  the  duct  is  the  chief  factor.  In- 
creased alkalinity  may  also  favor  precipitation  of  calcium  from  the  saliva.  In 
Roberg's  case  of  sialolithiasis  the  saliva  was  of  normal  composition. 

Intestinal  Concretions.— These  always  have  a  nucleus  of  some  indigestible 
foreign  substance,  most  often  hair,  but  sometimes  cellulose  structures  or  solid 
indigestible  particles,  including  gall-stones,  fruit-stones,  bone,  etc.  The  bulk  of 
the  concretions  is  usually  made  up  chiefly  of  ammonio-magnesium  phosphate,  with 
some  calcium  phosphate,  carbonate,  and  sulphate,  protein  matter,  and  occasionally 

'«  See  Lazarus,  Zeit.  klin.  Med.,  1904  (51),  530.     Literature. 
"  Rosenthal,  Arch.  f.  Verdauungskr.,  1914  (20),  619. 
"  Brit.  Med.  Jour.,  1896  (i),  1034. 
"  Lancet,  Dec.  18,  1909. 
"  Schmidt's  Jahrb.,  1900  (268),  210. 
«  Jour.  Pharm.  et  Chim.,  1901  (14),  21. 
'"Literature,  see  Roberg,  Annals  of  Surgery,  1904'(39),  t)69. 
"  Particles  of  gold  have  been  found  in  a  salivary  calculus  by  Maurin  (Repert 
pharm.,  1919  (30),  257),  presumably  derived  from  fillings. 
'8  Jour.  Pharm.  et  Chim.,  1903  (18),  11. 


INTESTINAL  CONCRETIONS  467 

calcium  and  magnesium  soaps.  Two  intestinal  concretions  analyzed  by  Schuberg" 
had  the  following  percentage  composition  when  dried : 

Ammonio-magnesiuni  phosphate 57. 1  63.9 

Calcium  i)hosphate If).?  23.8 

Calcium  carbonate 4.0 

Calcium  sulphate 3.0  0.7 

Alcohol-ether  extract 1.9  0.8 

Other  organic  substances 21 .5  6.0 

In  countries  where  oatmeal  is  largely  eaten,  intestinal  concretions  are  not  infre- 
quent; they  contain  calcium  and  magnesium  phosphate,  about  70  per  cent.; 
oatmeal  bran,  15-lS  per  cent.;  soaps  and  fats,  about  10  per  cent.  (Hammarsten). 
Occasionally  concretions  consisting  largely  of  fats  and  soaps  are  found,  and  after 
taking  large  doses  of  olive  oil  masses  of  solidified  oil  may  be  pa.ssed  that  are  readily 
mistaken  for  softened  gall-stones,  for  the  removal  of  which  the  oil  is  usually  given. 
The  "fecal  stones"  found  in  appendices  often  show  the  structure  of  calculi,  and, 
unlike  other  enteroliths,  consist  less  of  ammonio-magnesium  phosphate  than  of 
calcium  salts i^"  soaps  may  be  important  constituents.''^ 

Bezoar  stones  are  intestinal  concretions  probably  coming  from  Capra  aegngrus 
and  Antelope  dorcas.  One  variety  consists  chiefly  of  lithofellic  acid,  C2nH.-)fi04,  which 
is  related  to  cholalic  acid,  and  gives  an  aromatic  odor  when  heated.  The  other 
variety  ("false  bezoars")  does  not  give  the  aromatic  odor,  and  consists  chiefly 
of  ellagic  acid,  Ci4H60s,  a  derivative  of  gallic  acid,  and,  therefore,  probably  derived 
from  the  tannin  of  the  food  of  the  antelopes. 

Intestinal  "sand"  occurs  as  (1)  "false  sand,"  consisting  of  particles  of  indi- 
gestible food,  such  as  the  sclerenchymatous  particles  in  the  flesh  of  pears  and 
bananas;"  and  (2)  true  sand,  consisting  largely  of  inorganic  material,  and  formed, 
according  to  Duckworth  and  Garrod,^-^  in  the  upper  part  of  the  large  intestine. 
Analyses  of  specimens  by  Garrod  showed  the  following  composition: 

Water 12.4                        f  calcium  oxide 54 .  98 

Organic  material 26.29                      I  phosphorus  pentoxide 42.35 

Inorganic  material.  .  .      61  31  containing  |  carbon  dioxide 2.20 

[  traces  of  Mg,  Fe,  etc 0 .  47 

Analyses  by  other  observers  have  given  similar  results,  the  absence  of  the  large 
proportion  of  magnesium  found  in  larger  concretions  being  striking.  The  color 
is  usually  brown,  due  chieflj^  to  urobilin,  unaltered  bile-pigments  being  scanty. 

Preputial  concretions  sometimes  form  beneath  a  prepuce  that  cannot  be 
retracted,  through  deposition  of  urinary  salts  on  and  in  the  accumulated  smegma. ^^ 
The  composition  is,  therefore,  very  mixed,  and  consists  of  an  organic  base  contain- 
ing much  cholesterol,  fats,  and  soaps,  incrusted  with  inorganic  substances,  of 
which  ammonio-magnesium  phosphate  and  calcium  phosphate  are  usually  the 
most  abundant. 

Prostatic  concretions  originate  in  the  corpora  amylacea  through  growth  ac- 
cretion of  inorganic  salts,  until  they  may  reach  considerable  size.  Stern^'  gives 
the  following  results  of  analysis  of  such  a  prostatic  stone: 

Water 8.0 

Organic  matter.          .              .  15.8 

Lime ' 37 .  64 

Magnesia 2 .  38 

Soda 1 .  76 

Potash 0.5 

Phosphoric  acid 33 .  77 

Iron trace 

'9  Virchow's  Arch.,  1882  (90),  73. 

*°  Harlay,  Jour,  pharm.  et  chim.,  1910  (2),  433. 

^'  Williams,  Biochem.  Jour.,  1907  (2),  395. 

"  Myer  and  Cook,  Amer.  Jour.  Med.  Sci.,  1909  (137),  383. 

•"■^  Lancet,  1902  (i),  653.     Full  resume  and  literature. 

"  See  Zeller,  Arch.  klin.  Chir.,  1890  (41),  240. 

«  Amer.  Jour.  Med.  Sci.,  1903  (126),  281. 


468      CALCIFICATION,  CONCRETIONS,  AND  INCRUSTATIONS 


Lung  stones.*^ — These  may  be  formed  in  the  bronchi,  through  accretion  about 
an  inorganic  nucleus,  similar  to  the  formation  of  calculi  in  other  epithelial-lined 
passages;  or  they  may  consist  of  calcified  areas  of  lung  tissue  or  peribronchial 
glands,  which  have  been  sequestrated  through  suppuration  and  have  entered  the 
bronchi.  In  the  latter  case,  the  calculi  present  the  usual  composition  of  patho- 
logical calcified  areas.  That  the  expectorated  stones  frequently  represent  calcified 
tubercles  is  shown  by  Stern"  and  by  Biirgi."  who  demonstrated  tubercle  bacilli 
in  decalcified  lung  stones.     The  following  percentage  figures  are  taken  from  Ott:*' 

Calcium  phosphate 52 . 0  72 . 8 

Magnesium  phosphate 1.0 

Magnesium  carbonate 2.0 

Calcium  carbonate 13  0  6.0 

Fat  and  cholesterol 24 . 0  7.0 

Other  organic  substances 4.0  10.0 

Rhinoliths'^  are  formed  about  nasal  secretions,  blood-clots,  and  most  frequently 
about  foreign  bodies.  They  therefore  contain  much  organic  substance  in  addition 
to  the  inorganic  salts  deposited  upon  them  Berlioz"  gives  the  following  table 
from  the  analysis  of  four  specimens ; 


Weight  of  specimens,  grams 

1 
3.75 

2 
1.34 

3 
0.63 

4 
0.95 

Water 

Organic  matter 

Calcium  phosphate 

Magnesium  phosphate 

Calcium  carbonate 

Traces  of  iron 

5.80 
16.60 
62.02 

5.08 

10.50 

Doubtful. 

5.10 

18.20 

60.61 

6.28 

9.81 

Distinct. 

4.00 
16.00 
61.40 

3.93 
14.67 
Doubtful. 

6.90 

18.10 

47.63 

6.68 

20.69 

Distinct. 

Tonsillar  concretions  consist  chiefly  of  carbonate  and  phosphate  of  calcium 
deposited  upon  the  inspissated  secretions  and  desquamated  cells  of  the  tonsillar 
crypts.^"  According  to  some  authors,  leptothrix  threads  frequently  form  the 
nucleus  of  the  concretions. 

Cutaneous  concretions  are  occasionally  observed,  located  chiefly  in  the  sub- 
cutaneous tissue,  often  occurring  multiple.  The  origin  is  possibly  in  dilated 
sebaceous  glands  with  retained  secretions.  Unna  considers  that  calcium  soaps  are 
formed  as  a  first  step,  but  an  analysis  of  such  material  bj'  Harley^^  showed  87.2 
per  cent,  of  ash,  12.8  per  cent,  organic  matter,  0.9  per  cent,  of  fat;  calcium  phos- 
phate constituted  65.2  per  cent.,  and  calcium  carbonate  16.4  per  cent.  Gascard^^ 
found  in  similar  material  23.4  per  cent,  organic  matter,  and  of  the  inorganic  matter, 
91.1  per  cent,  was  calcium  phosphate,  and  8.9  per  cent,  calcium  carbonate. 

Gouty  deposits  observed  in  the  subcutaneous  tissues,  as  well  as  along  the 
tendons,  articular  cartilages,  etc.,  consist  usually  of  nearly  pure  biurate  of  sodium 
and  potassium.  Ebstein  and  Sprague^^  found  the  composition  of  such  material  to 
be  as  follows : 

Uric  acid 59 .  70 

Tissue  organic  matter 27 .  88 

Sodium  oxide 9 .  30 

Potassium  oxide 2 .  95 

Calcium  oxide 0.17 

MgO,  Fe,  P2O6,  S traces 

"Literature.  Poulalion,  Thesis,  Paris,  1891;  Stern,  Deut.  mcd.  Woch.,  1904, 
(30),  1414;  Burgi,  Deut.  med.  Woch.,  1906  (32),  798;  Gerhartz  and  Strigel, 
Beitr.  z.  klin.  Tubarc,  1908  (10),  33. 

*''  "Chem.  Path,  der  Tuberc,"  1903,  p.  92. 

■»8  Literature,  Scheppegroll,  Jour.  Amer.  Mcd.  Assoc,  1896  (20),  874;  Gcrber, 
Deut.  med   Woch.,  1892  (18),  1165. 

*^  Jour.  Pluirm.  et  Chiin.,  1891   (23),  447. 

^"McCarthy,  Brit.Med.  Jour.,  Oct.  28,  1911. 

'''  Jour.  Pliarm.  et  Chim.,  1903  (18),  9. 

»2  Ibid.,  1900  (12),  262. 

"  Virchow's  Arch.,  1891  (125),  207. 


PULMONARY  INCRUSTATIONS  400 

After  a  time,  however,  calcium  salts  may  be  deposited,  and  Dunin"  has  observed 
deposits  resembling  gouty  tophi  that  were  merely  calcium  salts. 

Pneumonokoniosis 

In  a  number  of  cases  of  the  different  forms  of  this  con(htion  quan- 
titative anal3'-ses  have  been  made,  which  may  be  briefly  discussed  as 
follows:  Not  only  docs  the  lung  of  every  adult  contain  considerable 
amounts  of  coal-pigment  stored  up  in  the  connective  tissues  (and  also 
in  the  peribronchial  glands),  but  also,  which  is  perhaps  less  generally 
appreciated,  considerable  quantities  of  sihcates  are  also  present  (chal- 
icosis)  from  inhaled  dust.  Woskressensky^^  found  silicates  in  all  of 
54  lungs  examined,  except  two  from  infants.  The  lungs  of  individ- 
uals whose  occupations  do  not  expose  them  especially  to  dust  inhala- 
tion contain  increasing  amounts  of  silicates  in  direct  proportion  to 
age;  the  silicates  constitute  then  from  3.5  to  10  per  cent,  of  the  total 
ash  of  the  lungs.  There  is  always  a  larger  proportion  of  silicates 
in  the  peribronchial  glands  than  in  the  lungs,  constituting  from  6  to 
36  per  cent,  of  the  ash,  corresponding  with  Arnold's  observation  that 
in  gold-beaters  the  glands  contain  more  metal  than  the  lungs.  In 
stone-workers  Schmidt  found  a  higher  proportion  of  Si02  in  the  lungs 
than  in  the  glands.  In  normal  adults  the  amount  of  coal-pigment 
is  greater  than  the  amount  of  silicates;  in  children  the  reverse  is  the 
case. 

ThoreP®  reports  that  the  lungs  of  a  worker  in  soapstone  contained 
3.25  per  cent,  of  ash,  including  2.43  per  cent,  of  soapstone. 

In  siderosis  iron  has  been  found  in  the  lungs  in  proportions  varying 
from  0.5  per  cent,  to  7.9  per  cent,  of  the  dry  weight,  the  last  amount 
having  been  found  by  Langguth"  in  the  lungs  of  an  iron  miner,  which 
contained  also  11.92  per  cent,  of  Si02. 

An  analysis  of  a  lung  from  a  knife-grinder  is  reported  b}''  Hoden- 
pyl^ss  which  gave  the  following  results:  Total  weight  of  dried  and 
powdered  lung,  48.1009  grams;  total  solids,  44.7986;  ether-soluble 
substance,  14.6017.  Composition  of  the  ether-soluble  substance:  free 
fatty  acids,  7.498;  neutral  fats,  4.044;  cholesterol,  3.037.  Proteins, 
15.4759;  charcoal  (total  carbon  less  protein  carbon),  7.198;  ash, 
4.2903.  The  composition  of  the  ash  (in  grams)  was  as  follows:  K2O, 
0.2167;  NaaO,  0.3523;  CaO,  0.0965;  Fe20„  0.0879;  AI2O3,  1.4628; 
SO3,  0.0704;  P2O5,  0.9565;  Si02,  1.2043.  The  amount  of  emery,  rep- 
resented by  the  oxides  of  aluminum  and  silicon  made  up  more  than 
one-half  of  the  ash,  and  the  iron  constituted  about  one-fourth.  The 
man  had  worked  at  the  trade  of  knife-grinder  for  about  fifteen  years. 

"  Mitt.  Grenzgeb.  Med.  u.  Chir.,  1905  (14),  451;  also  Kahn,  Arch.  Int.  Med.^ 
1913  (11),  92,  and  M.  B.  Schmidt,  Deut.  med.  Woch.,  1913  (39),  59. 
"  C^.ot.  f.  Path..  1898  (9),  296. 
58  Ziegler's  Beitr.,  1896  (20),  85. 
"  Deut.  Arch.  klin.  Med.,  1895  (55),  255. 
'«  Medical  Record,  1899  (56),  942. 


470     CALCIFICATION,   CONCRETIONS,   AND  INCRUSTATIONS 

McCrae^^  has  analyzed  the  lungs  of  six  gold  mine  workers,  in  South 
Africa,  finding  from  9  to  21.7  grams  of  ash  per  lung,  of  which  29  to 
48  per  cent,  was  silica;  aluminum  was  also  high,  and  an  increased 
PoOs  content  was  ascribed  to  the  accompanying  fibrosis.  Klotz^° 
found  from  1.2  to  5.3  grams  of  free  carbon  in  each  lung,  of  dwellers 
of  Pittsburg,  as  contrasted  with  0.145  and  0.405  grams  found  in  the 
lungs  of  residents  of  Ann  Arbor.  Hirsch^^  analyzed  four  average 
Chicago  lungs,  finding  in  grams  per  lung: 

I  II  III              IV 

Carbon 2.72  0.71  1.20  0.19 

Silica 0.18  0.28  0.69  0.04 

Calcium  Oxide 0.45  0.12  0.02  0.05 

^5  "The  Ash  of  Silicotic  Lungs,"  John  McCrae,  Johannesburg,  1914. 

^0  Aineh  Jour.  Publ.  Health,  1914  (4),  887.     General  review  on  anthracosis. 

"  Jour.  Amer.  Med.  Assoc,  1916  (66),  950 


CHAPTER  XVIII 

PATHOLOGICAL  PIGMENTATION' 

MELANIN^ 

Melanin  occurs  normally  as  the  coloring-matter  of  hair,  of  the 
choroid  of  the  eye,  of  the  skin,  in  the  pigment  matter  of  many  lower 
animals,  and  most  strikingly  as  a  defensive  substance  in  the  "ink'" 
ejected  by  squids  to  render  themselves  invisible  in  the  water.  Path- 
ologically melanin  occurs  chiefly  as  the  result  of  an  excessive  pro- 
duction of  this  pigment  by  cells  normally  forming  it,  as  in  freckles, 
melanotic  tumors,  and  Addison's  disease  (probably).  Cells  that  do 
not  normally  form  melanin  probably  do  not  acquire  this  power  in 
pathological  conditions.  Pathological  failure  to  form  melanin  is  also 
observed,  as  in  skin  formed  in  the  healing  of  wounds  and  after  syphili- 
tic lesions;  or  in  albinis77i,  in  w'hich  the  failure  to  form  melanin  may  be 
attributed  to  hereditary  influences.^  Occasionally  in  domestic 
animals,  especially  in  calves,  a  congenital  melanosis  is  observed  in- 
volving many  parts  of  the  body."*  A  melanin  or  some  similar  pig- 
ment may  be  found  in  nerve  cells  (e.  g.,  substantia  nigra),  and  DoUey* 
beheves  it  to  be  a  result  of  nuclear  metabolism  under  conditions  of 
depression.  The  function  of  melanin  is  evidently  that  of  protection 
from  light  rays,  and  Young*'  has  found  that  isolated  melanin  from  hu- 
man skin  absorbs  violet  and  ultra-violet  rays.  Probably  this  protec- 
tion is  responsible,  at  least  in  part,  for  the  relative  infrequency  of  skin 
cancers  in  the  colored  races.'' 

Melanin  seems  always  to  be  produced  through  metabolic  acti\'ity 
of  specialized  cells.  The  idea,  which  was  formerly  advanced,  that 
it  is  derived  from  hemoglobin  as  a  product  of  disintegration,  seems 
to  have  failed  entirely  of  substantiation.  In  malaria  we  frequently 
find  a  diffuse  pigmentation  of  the  skin  of  such  a  nature  as  to  suggest 

'  Literature  by  Oberndorfer,  Ergebnisse  Pathol.,  1908  (12),  460,  and  Hueck, 
Ziegler's  Beitr.,  1912  (54),  68. 

2  Literature  and  resume  given  by  v.  Ftirth,  Cent.  f.  Pathol.,  1904  (15),  617; 
Handb.  d.  Biochem.,  1,  742. 

'  Gortner  holds  that  dominant  whites  are  due  to  the  presence  of  antioxidase, 
while  regressive  whites  have  neither  the  power  to  form  pigments  nor  to  inhibit 
their  formation  (Amer.  Naturalist,  1910   (44),  497). 

*  See  Caspar,  Ergebnisse  allg.  Path.,  1896  (III2),  772. 

'  Science,  1919  (50),  190. 

«  Biochem.  Jour.,  1914  (8),  460. 

'  However,  Hanawa  found  white  areas  in  skin  less  affected  by  chemical  irri- 
tants and  infections  than  dark  areas.  (Dermatol.  Zeit.,  1913  (20),  761.)  This  is 
not  in  agreement  with  most  observers  who  have  found  pigmented  skin  more  re- 
sistant.     (See  Hanzlik  and  Tarr,  Jour.  Pharm..  1919  (14),  221.) 

471 


i 


472  PATHOLOGICAL  PIGMENTATION 

strongly  a  melanin  formation,  and  this  has  been  cited  as  an  example 
of  the  production  of  melanin  from  hemoglobin.  Carbone  has  proved, 
however,  that  this  malarial  pigment  is  derived  from  hematin.  The 
amount  of  iron  contained  in  melanin  has  been  much  investigated,  as 
bearing  upon  the  question  as  to  whether  the  melanin  is  derived  from 
hemoglobin  or  not,  and  the  results  obtained  by  the  best  methods  indi- 
cate that  the  amount  of  iron  present  is  usually  extremely  small,  and 
often  it  is  entirely  absent;  furthermore,  the  presence  of  iron  is  no 
proof  that  the  pigment  is  derived  from  hemoglobin,  since  other  iron- 
protein  compounds  undoubtedly  exist,^ — especially  nucleoproteins, 
and  chemical  examination  shows  that  melanin  does  not  contain  hemo- 
pyrrole  groups.^ 

Composition  of  Melanin. — The  elementary  composition  of  different  specimens 
of  melanin  examined  by  various  observers  has  been  found  to  vary  greatly.  This 
probably  depends  on  three  factors :  First,  it  is  extremely  difficult  to  obtain  melanin 
in  a  pure  condition;  second,  the  process  of  purification  requires  the  action  of  strong 
acids  and  alkalies,  which  undoubtedly  modify  the  composition  of  the  melanin; 
thirdlj^,  melanin  is  probably  not  a  single  substance  of  definite  composition,  but 
includes  several  related  biit  different  bodies.  The  values  found  varj'  for  carbon 
from  48.95  to  60.02  per  cent. ;  for  hydrogen  from  3.05  to  7.57  per  cent. ;  for  nitrogen, 
8.1  to  13.77  per  cent.  Hofmeister  gives,  as  a  characteristic  of  melanins,  that 
their  elementary  molecular  composition  is  always  nearly  in  the  proportions 
N  :  H  :  C  =  1  :  5  :  5. 

Gortner's^  studies  have  led  him  to  accept  the  general  principle  that  melanin 
is  formed  through  the  action  of  an  oxidase  on  an  o.xidizable  chromogen,  but  that 
in  keratinous  structures  there  exist  at  least  two  types  of  melanins,  one,  a  "nielano- 
protein,"  soluble  in  dilute  acids  and  existing  dissolved  in  the  keratins;  the  other, 
insoluble  in  dilute  acids,  exists  as  pigment  granules  and  is  of  unknown  nature. 
Piettre'"*  believes  that  melanin  from  sarcoma  of  the  horse  consists  of  a  protein 
united  to  a  pigment.  Those  whose  studies  of  melanin  formation  have  been  made 
with  the  microscope,  state  that  the  nucleus  is  active  in  the  process, ^^  and  some 
find  the  melanin  so  closely  related  to  the  lipoids  that  they  consider  it  a  lipochrome.^^ 

A  particularly  prominent  constituent  of  some  melanins  is  sulphur,which  has 
been  found  in  as  high  proportions  as  10  per  cent,  in  melanin  from  sarcomas,  and 
even  12  per  cent,  in  sepia  from  the  squid;  in  melanin  from  hair  the  sulphur  is 
usually  about  2-4  per  cent.;  but  in  choroid  melanin,  and  in  some  other  forms, 
sulphur  seems  to  be  absent.  The  proportions  of  sulphur  obtained  from  the  same 
specimen  purified  by  different  methods  show  wide  variations,  and  hence  v.  Fiirth 
considers  that  neither  the  sulphur  nor  the  iron  are  indispensable  constituents  of 
the  melanin.  Probably  the  melanin  molecule  contains  atom-complexes  that  have 
a  tendency  to  bind  certain  sulphur  and  iron  compounds  (e.  g.,  cystine  or  hematin 
derivatives). 

There  is  much  reason  to  believe  that  the  melanin  is  derived  from  certain  groups 
of  the  protein  molecule  that  seem  readily  to  form  colored  comjiounds.  The  aro- 
matic compounds  of  the  protein  molecule,  such  as  tyrosine,  phenylalanine,  and 
tryptophane,  readily  condense  with  elimination  of  water  and  absorption  of  oxygen, 
to  produce  dark-colored  substances.  When  proteins  are  heated  in  strong  hydro- 
chloric acid,  we  obtain  a  dark-brown  material,  which  closely  resembles  the  melanins 
both  in  elementary  composition  and  in  general  properties,  so  that  it  is  referred  to 

8  Spiegler,  Hofmeister's  Beitr.,  1907  (10),  253. 
»Biochem.  Bulletin,  1911  (1),  207;  r6sum6. 

*"  Compt.  Rend.  Acad.  Sci.,  1911  (153),  782;  also  see  Reprint  from  1st  Internat. 
Cong.  Compar.  Pathol.,  Paris,  1912. 

1'  Htaffel,  Verh.  Deut.  Path.  Ges.,  1907  (11),  136;  Schultz,  Jour.  Med.  Res., 
1912  (26),  65. 

12  Dyson,  Jour.  Path,  and  Bact.,  1911  (15),  298;  Kreibich,  Wien.  klin.  Woch., 
1911  (24),  117. 


MELANIN  473 

as  "artificial  melanin"  or  "melanoid  substance."  These  substances,  like  the 
natural  inelanins,  when  decomposed  by  fusing  with  caustic  potash,  yiehl  skatolc, 
indole,  and  pyrrole  derivatives,  whicli  are  undoubtetily  derived  from  the 
tyrosine  and  tryptophane  of  the  protein  molecule.  Therefore,  it  seems  probable 
that  both  the  melanoid  substances  and  the  true  melanins  are  formed  from  the 
chromojfen  groups  of  the  protein  molecule  through  processes  of  condensation, 
elimination  of  water,  and  the  taking  up  of  oxygen.'* 

In  the  sepia  sacs  of  the  cuttle-fish,  in  meal-worms  which  form  a  melanin-like 
pigment,  and  in  plants  that  produce  the  black  Japanese  lacquer,  have  been  found 
oxidizinq  enzymes  that  have  the  property  of  producing  black  pigment  by  their 
action  upon  tyrosine  and  other  aromatic  compounds.  Neuberg"  found  that  ex- 
tracts of  a  melanosarcoma  of  the  adrenal  could  produce  pigment  from  epinephrine 
and  /3-oxyphenylethylamine,  but  not  from  tyrosine.  The  ink  sacs  of  the  squid 
contain  an  enzyme  forming  a  pigment  from  epinephrine,  apparently  through  oxi- 
dation and  condensation.  These  enzymes  may,  therefore,  possibly  be  responsible 
for  the  production  of  melanin  in  animal  tissues,  by  causing  oxidative  changes  in 
the  chromogen  groups  of  the  protein  molecule  that  are  liberated  by  autolysis  (see 
"Tyrosinase"),  v.  Ftirth  urges  strongly  the  view  that  both  normal  and  patho- 
logical melanin  formation  depend  upon  the  action  of  the  tyrosinase  or  allied  en- 
zymes in  conjunction  with  autolytic  enzymes;  the  latter  split  free  the  chromogen 
groups  of  the  protein  molecule,  which  are  then  oxidized  by  the  tyrosinase,  undergo 
condensation,  and  take  up  sulphur-  and  iron-holding  groups  and  also  other  organic 
compounds,  the  entire  complex  forming  the  melanin. 

Bruno  Bloch'^  has  found  that  the  occurrence  of  melanin  in  the  skin  corresponds 
to  the  location  of  cells  with  the  capacity  of  oxidizing  3.4-dioxyphenylalanine, 
which  is  closely  related  in  structure  to  epinephrine,  and  which  he  believes  may  be 
the  usual  antecedent  of  melanin.  He  has  found  this  oxidizing  property  exhibited 
by  the  dark  patches  in  variegated  animals,  but  not  by  the  white  areas;  the  pig- 
mented ocular  structures  do  not  oxidize  this  substance. 

Properties  of  Melanin. — When  isolated  in  a  pure  condition, 
melanin  is  a  dark-brown  substance  of  amorphous  structure,  no  mat- 
ter how  black  the  material  from  which  it  is  derived  may  be.**'  It  is 
quite  insoluble  in  all  ordinary  reagents  except  alkalies,  in  which  some 
melanins  dissolve  easily,  and  some  with  difficult3\  Strong  boiling 
hydrochloric  acid  scarcely  affects  non-protein  melanins.  By  the 
action  of  sunhght  or  oxichzing  agents  on  melanin-containing  sections 
the  pigment  can  be  bleached  out.  The  chief  decomposition-products 
formed  on  fusing  with  alkalies  are  indole,  skatole,  and  "melanic 
acid";  no  cystine,  leucine,  tyrosine,  or  other  amino-acids  can  be  iso- 
lated. Most  authors,  therefore,  consider  the  melanins  as  heterocyclic 
compounds  standing  in  some  relation  to  the  indole  nucleus. 

If  melanin  is  injected  subcutaneously  into  animals  u'abbits  and 
guinea-pigs),  there  appears  in  the  urine  a  substance  which  turns  dark 
brown  after  the  urine  has  stood  for  some  time  (Kobert,  Helman). 
The  pigment  is  apparently  reduced,  particularly  by  the  liver,  to  a 
colorless  melanogen,  which  is  eliminated  in  the  urine.  The  same 
process  occurs  when  melanin  is  produced  in  excess  and  enters  the 

'3  See  Herzmark  and  von  Furth,  Biochem.  Zeit.,  1913  (49),  130. 

1*  Zeit.  f.  Krebsforsch.,  1909  (8),  195. 

15  Bloch  and  Ryhiner,  Zeit.  exp.  Med.,  1917  (5),  179;  Zeit.  physiol.  Chem., 
1917  (100),  226. 

'^  Spiegler  (Hofmeister's  Beitr.,  1903  (4),  40)  claims  to  have  isolated  from 
white  wool  a  white  chromogen,  closely  related  to  melanin  chemically,  but  Gortner 
(Amer.  Naturalist,  1910  (44),  497)  believes  this  to  be  a  decomposition  product  of 
keratin,  unrelated  to  melanin. 


474  PATHOLOGICAL  PIGMENTATION 

blood,  as  in  the  case  of  melanosarcoma,  a  colorless  melanogen  being 
formed  which  is  excreted  in  the  urine,  constituting  "melanuria." 
Occasionally  the  urine  is  dark  when  first  passed,  because  of  the  pres- 
ence of  melanin,  but  usually  it  must  be  subjected  to  oxidizing  agen- 
cies (^bromine  water,  nitric  acid,  hypochlorites,  etc.),  or  exposed  to 
air  to  bring  out  the  brown  color.  Helman^^  says  that  true  melano- 
gen may  be  considered  to  be  present  in  urine:  \1)  If  the  careful  ad- 
dition of  ferric  chloride  causes  the  development  of  a  black  precipi- 
tate. (2)  If  this  precipitate  dissolves  in  sodium  carbonate,  forming 
a  black  solution.  (3)  If  from  this  solution  mineral  acids  precipitate 
a  black  or  brownish-black  powder.  All  three  reactions  must  be 
obtained,  for  substances  other  than  melanin  may  give  the  first  two. 
Especially  to  be  distinguished  are  alkaptonuria,  chronic  intoxication 
with  phenols,  and  some  cases  of  extreme  indicanuria.^*  In  support  of 
the  view  that  tryptophane  is  the  mother  substance  of  melanin  is  the 
fact  that  feeding  tryptophane  to  melanurics  increases  the  melanin 
excretion  (Eppinger). 

The  coloring  power  of  melanin  is  very  great,  for  urine  containing 
but  0.1  per  cent,  of  melanin  has  the  color  of  dark  beer  (Hensen  and 
Nolke),  and  the  entire  skin  of  a  negro  contains  only  about  1  gram 
of  melanin  (Abel  and  Davis). '^  Excessive  quantities  of  melanin  may 
be  in  part  deposited  in  the  lymph-glands  and  skin,  causing  diffuse 
pigmentation;  it  may  be  deposited  in  the  endothelium  lining  the 
blood-vessels.  Koberfc  injected  melanin  into  albino  rabbits,  but 
did  not  succeed  in  getting  any  deposition  in  the  choroid  or  skin. 
Helman  found  some  evidence  of  toxicity  when  large  doses  of  melanin 
dissolved  in  sodium  carbonate  are  injected  into  animals,  but  this  is 
possibly  due  to  the  alkali  rather  than  to  the  melanin. 

Melanotic  Tumors.^" — Tumor  melanin  does  not  differ  from  mel- 
anin produced  by  normal  cells  in  any  essential  respect.  Usually  it  con- 
tains much  sulphur,  even  as  much  as  10  per  cent.,  yet  Helman  in  eight 
specimens  found  but  four  that  contained  both  sulphur  and  iron,  in 
three  only  sulphur,  in  one  only  iron  and  no  sulphur;  therefore,  tumor 
melanins  show  the  same  variations  in  composition  as  do  normal  mel- 
anins.  Iron  is  frequently  found  microscopicallj'-  in  the  pigment  in 
melanosarcoma,  but  this  is  chiefly  due  to  admixture  of  blood-pigment 
coming  from  extravasations  of  blood.  The  peculiar  fact  that  melano- 
sarcoma is  very  common  in  white  or  gray  horses,  but  very  seldom 

"  Cent.  f.  inn.  Med.,  1902  (23),  1017;  Arch,  internat.  Pharmakodynam.,  1903 
(12),  271. 

^8  Melanuria  fully  discussed  by  Feigl  and  Querner,  Deut.  Arch.  klin.  Med., 
1917(123),  107. 

"  Jour.  Exp.  Med..  189G  (1),  3G1. 

2"  Under  the  title  Acanthosis  Nigi'icans  (see  PoUitzer,  Jour.  Amer.  Med.  Assoc, 
1909  (53),  1369)  is  included  a  group  of  cases  of  widespread  cutaneous  pigmentation 
with  papillary  hypertrophy,  commonly  associated  with  cancer,  most  often  ab- 
dominal. While  ascribed  to  action  of  the  sympathetic  nervous  system  injured 
by  the  cancer,  this  explanation  is  far  from  satisfactory,  and  the  possibility  of 
metabolic  pigmentary  disturbance  must  be  considered. 


MELANIN  475 

occurs  in  dark-coated  horses,  has  not  been  explained.  The  frequent 
occurrence  of  mehmuria  and  mehincniia  in  patients  witli  iiiolanosar- 
coma  is  not  duo  to  any  peculiar  property  of  sarcoma  nichmin,  but  to 
the  enormous  quantity  of  melanin  that  is  prochKuul  by  the  tumor  and 
set  free  in  the  degenerating;  portions.  Thus,  while  Abel  and  Davis'^ 
estimate  that  there  is  only  about  1  gram  of  melanin  in  the  entire  skin 
of  a  negro,  Nencki  and  Bordez  have  obtained  from  a  sarc-omatous 
liver  300  grams  of  melanin,  and  estimate  that  the  entire  body  con- 
tained 500  grams.  Helman'^  states  that  the  melanin  may  con- 
stitute 7.3  per  cent,  by  weight  of  the  fresh  substance  of  some 
melanosarcomas.  According  to  Lubarsch  and  to  Helman,  melanotic 
tumors  rarely  contain  glycogen. 

As  mentioned  above,  Neubcrg  found  that  a  melanotic  sarcoma  of 
the  adrenal  produced  pigment  from  epinephrin  and  from  /3-oxy- 
phcnylethylamine,  but  he  failed  to  get  positive  results  with  melano- 
sarcomas of  the  eye  and  from  the  horse,  but  Alsberg-'  succeeded  in 
finding  in  melanosarcoma  from  the  liver  an  enzyme  oxidizing  pyro- 
catechin  and  Jager-  found  that  horse  melanosarcoma  extracts  will 
oxidize  epinephrin  to  a  pigment.  The  "dopa  reaction"  of  Bloch,^^ 
which  depends  on  the  presence  of  specific  oxidizing  enzymes  in  the 
cells,  may  be  exhibited  by  the  connective  tissues  quite  generally 
throughout  the  body  in  some  cases  of  melanosarcoma."" 

Eppinger-^  found  that  the  urine  of  a  patient  with  melanosarcoma 
gave  intense  reactions  for  indole  and  tryptophane,  and  that  when 
tryptophane  was  fed  to  a  patient  there  was  a  great  increase  in  the 
melanuria.  He  therefore  concludes  that  the  power  of  the  body  to 
destroy  the  pyrrole  ring  is  reduced,  and  instead  it  undergoes  reduc- 
tion, methylation  and  union  with  sulphuric  acid,  to  form  an  ethereal 
sulphate  of  methylpyrrolidine-hydroxy-carbonic  acid  (CH3-C5H9N2O4). 
Abderhalden-''  also  found  a  relation  to  tryptophane,  for  in  the  urine 
of  a  melanuric  was  present  a  substance  rich  in  tryptophane;  and 
Primavera"  found  the  urine  in  a  case  of  melanosarcoma  containing 
free  tyrosine,  fluctuating  in  amount  with  the  pigment. 

Addison's  disease  is  associated  with  the  deposition  of  a  pigment 
in  the  skin  that  is  generally  considered  to  be  a  melanin,  differing 
from  that  produced  normally  in  the  skin  only  in  quantity  and  not  in 
origin  or  composition. ^"^  No  satisfactory  explanation  of  the  relation 
of  the  adrenal  to  this  pigmentation  seems  yet  to  have  been  made,  al- 
though it  is  natural  to  assume  that  when  the  function  of  the  adrenal 
is  destroyed,  substances  accumulate  in  the  blood  that  have  a  stimu- 
li Jour.  Med.  Res.,  1907  (16),  117. 

22  Virchow's  Arch.,  1909  (198),  62. 

•""  Matsunaga,  Frankf.  Zeit.  Path.,  1919  (22),  69. 

"  Biochem.  Zeit.,  1910  (28 j,  181. 

2^  Zeit.  physiol.  Chem.,  1912  (78),  159. 

"  Giorn.  Int.  Scienze  Med.,  1908  (29),  978. 

-«  Concerning  histogenesis  of  the  pigment  see  Pforringer,  Cent.  f.  Path.,  1900 
(11),  1. 


476  PATHOLOGICAL  PIGMENTATION 

lating  effect  on  the  pigment-forming  cells.  Abnormal  protein  catab- 
olism,  with  excessive  accumulation  of  the  chromogenic  constituents  of 
the  protein  molecule,  has  been  suggested,  as  also  have  alterations  in 
the  influence  of  the  sympathetic  nervous  system  upon  the  chromo- 
phore  cells,  for  nerve  lesions  (e.  g.,  neurofibroma)  often  are  accom- 
panied by  pathological  pigmentation  of  the  skin.^^ 

It  is  significant  that  the  active  constituent  of  the  adrenal  medulla, 
the  epinephrin,  is  an  aromatic  derivative  closely  related  to  tyrosine, 
since  the  production  of  pigment  by  the  action  of  oxidizing  enzymes 
upon  such  substances  is  well  known.  Furthermore,  Neuberg  has 
described  a  melanotic  adrenal  tumor  which  produced  pigment  by 
oxidizing  epinephrine.  On  this  basis  the  pigmentation  of  Addison's 
disease  would  seem  to  be  the  result  of  an  abnormal  accumulation  or 
distribution  of  aromatic  compounds,  because  of  their  failure  to  be 
converted  into  epinephrine.  In  support  of  this  hypothesis  is  the 
observation  of  Meirowsky  that  the  human  skin  contains  an  enzyme 
capable  of  oxidizing  epinephrine  to  a  pigment,  and  that  pieces  of 
skin  kept  warm  will  develop  a  postmortem  pigmentation,  and  this  is 
supported  by  Konigstein-^  who  found  that  the  pigmentation  was 
greater  in  animals  deprived  of  their  adrenals  or  given  injections  of 
epinephrine.  Bloch^^  believes  that  the  pigmentation  results  from  the 
precursor  of  epinephrine,  3.4-dioxyphenylalanine,  which  is  oxidized 
in  the  epidermal  cells  to  melanin. 

As  exact  chemical  studies  of  the  pigment  in  Addison's  disease  have 
not  been  made,  however,  we  have  no  positive  proof  that  it  is  a  mel- 
anin, hence  any  speculation  as  to  the  cause  of  its  formation  is  prema- 
ture. Carbone-^  claims  to  have  isolated  from  the  urine  in  Addison's 
disease  a  pigment  that  contains  much  sulphur,  and  which  he  considers 
similar  to  or  identical  with  the  melanogen  of  melanuria.  A  similar 
observation  is  reported  by  Eiselt.^''  v.  Kahlden,^^  however,  has  ob- 
served crystals  resembling  hematoidin  in  the  pigmented  tissues. 

Ochronosis'^  is  a  condition  characterized  bj^  a  black  pigmentation 
of  the  cartilages,  first  described  by  Virchow  in  1866.  In  1904  Osler'^ 
reported  two  cases,  and  found  but  seven  others  in  the  literature  to  that 
time.  Virchow  suspected  that  the  condition  was  due  to  a  permeation 
of  cartilage  by  hematin  derivatives,  but  Hansemann,  finding  a  case 
associated  with  melanuria,  considered  that  the  pigment  is  probably  of 
metabohc  origin.  Hecker  and  Wolf  studied  the  urine  of  a  similar 
case,  and  concluded  that  the  pigment  must  be  melanin.     Albrecht,^* 

"  See  r6s\im6  by  Schmidt,  Ergeh.  der  Pathol.,  1896  (Bd.  3,  Abt.  1),  551. 

"  Wien.  klin.  Woch.,  1910  (23),  616. 

29  Giorno  R.  Acad.  med.  di  Torino,  1896. 

'"  Zeit.  klin.  Med.,  1910  (69),  393;  full  discussion  on  the  pigment  of  Addison's 

dlSGA-SG 

»iVirchow's  Arch.,  1888  (114),  65. 

32  See  Adler,  Zeit.  f.  Krebsforsch.,  1911  (11),  1;  Poulsen,  Ziegler's  Beitr.,  1910 
(48),  346. 

"Lancet,  1904  (i),  10  (literature). 

»<  Zeit.  f.  Heilk.,  Path.  Abt.,  1902  (23),  366. 


I 


OCHRONOSIS  471 

however,  suggested  a  relation  of  ochronosis  to  alkaptonuria,  having 
found  honiogentisic  acid  in  the  urine  of  a  case  reported  by  him  (see 
"Alkaptonuria")-  Osier's  two  patients  were  brothers  with  alkap- 
tonuria, the  evidence  of  ochronosis  consisting  of  discoloration  of 
the  cartilages  of  the  ears.  Langstein'"'  has  examined  a  specimen  of 
urine  preserved  from  Hansemann's  case,  and  found  no  evidence  of 
alkaptonuria.^*^ 

Pick"  summarizes  the  results  of  his  study  of  his  case  and  of  the 
literature,  as  follows:  Ochronosis  is  a  definite  form  of  melanotic  pig- 
mentation, the  pigment  of  ochronosis  being  in  most  of  the  cases  very 
closely  related  to  melanin.  The  pigment,  or  its  chromogcn,  circulating 
freely  in  the  blood,  is  imbibed  not  only  by  cartilage,  but  also  by  loose 
connective  tissue,  voluntary  and  involuntary  muscle-cells,  and  epi- 
thelial cells,  without  any  decrease  in  vitality  of  these  cells  being 
observable;  however,  degenerated  tissues  show  the  greatest  amount  of 
pigmentation.  The  diffuse  pigment  can  become  granular  after  a  time; 
it  is  iron-free,  but  under  certain  circumstances  may  contain  fat.  This 
melanin  arises  from  the  aromatic  nucleus  of  the  protein  molecule  (tyrosine, 
phenylalanine),  and  the  related  hydroxylized  products,  under  the 
influence  of  tyrosinase.  In  some  cases  the  constant  absorption  of  mi- 
nute quantities  of  phenol  from  surgical  dressings  seems  to  have  been  the 
cause  of  the  condition.  Besides  this  formation  of  pigment  from  such 
"exogenous"  aromatic  substances,  however,  it  is  probable  that  in 
alkaptonuria  the  "endogenous"  aromatic  substance  (homogentisic 
acid)  present  may  be  converted  into  pigment  by  the  tyrosinase. 
In  many  of  the  cases  of  ochronosis  the  pigment  or  a  precursor  may  be 
excreted  in  the  urine,  which  then  undergoes  spontaneous  darkening 
when  exposed  to  the  air.  The  kidneys  may  also  become  pigmented 
and  granular  masses  of  pigment  may  be  present  in  the  renal  tubules. 

Poulsen^*^  states  that  of  the  32  known  cases  of  ochronosis  (in  1911) 
in  17  there  was  alkaptonuria,  in  8  carbohc  acid  dressings  had  been 
used  for  long  periods,  and  in  the  remaining  7  cases  the  cause  was  not 
determined.  These  facts  are  conclusive  evidence  of  the  origin  of 
ochronotic  pigment  from  aromatic  radicals,  whether  these  radicals 
are  converted  into  true  melanin  or  not.  The  localization  of  the  pig- 
ment is  explained  by  the  demonstration  by  Gross  and  Allard,^^  that 
cartilage  has  a  greater  affinity  than  other  tissues  for  homogentisic 
acid.  Ochronosis  can  be  produced  experimentally  with  homogentisic 
acid,  and  often  is  associated  with  an  arthritis. •*"  There  are,  however, 
numerous  cases  of  alkaptonuria  without  ochronosis.  The  ochronosis 
described  in  lower  animals  is  not  the  same  as  human  ochronosis,  affect- 

35  Hofmeister's  Beitr.,  1903,  (4)145. 

3«  Also  see  Langstein,  Berl.  klin.  Woch.,  1906  (43),  597. 

"  Berl.  klin.  Wochenschr.,  1906  (43),  478. 

38  Mlinch.  med.  Woch.,  1912  (59),  364. 

33  Arch.  exp.  Path.  u.  Pharm.,  1908  (59),  384. 

"  Gross,  Deut.  Arch.  klin.  Med.,  1919  (128;,  249. 


478  PATHOLOGICAL  PIGMENTATION 

ing  the  bones  rather  than  the  cartilages  (Poulsen),-'^  and  being  more 
properly  designated   by  the  name  osteohemachromatosis  (^Schmey).''^ 

Malarial  pigmentation,  according  to  Ewing/'  may  have  any  one  of  the  follow- 
ing origins: 

(1)  Pigment  elaborated  by  the  intracellular  parasite.  (2)  Hematoidin  de- 
rived from  the  remnants  of  infected  red  cells.  (3)  Hematoidin  or  altered  hemo- 
globin deposited  in  granular  or  crj'stalline  form  from  red  cells  dissolved  in  the 
plasma.     (4)  Bilirubin  or  urobilin  granules  or  crystals. 

Of  these,  the  pigment  formed  by  the  parasites  has  been  considered  by  many 
as  a  true  melanin,  but  this  cannot  be  considered  as  established,  especially  as  Ewing 
finds  it  to  have  the  same  relation  to  solvents  as  do  the  blood-pigments.  Carbone 
and  Brown^^  consider  the  malarial  pigment  to  originate  from  hematin,  with  which 
it  agrees  in  solubility,  spectroscopic  properties,  and  in  containing  iron. 

Pigmentation  of  the  Colon.'*-^ — Sometimes  the  mucosa  of  the  entire  colon  is 
found  deeply  pigmented,  with  a  material  of  unknown  character,  but  resembling 
in  many  respects  a  melanin.  The  cause  of  the  condition  is  unknown.  Abder- 
halden"  has  found  pigments  that  seemed  to  be  derived  from  tryptophane,  while 
Niklas^^  attributes  the  coloration  to  tyrosinase  activity. 

Pigmentation  of  the  oral  mucosa,  with  a  pigment  resembling  melanin,  has  been 
described  especiallj^  in  pernicious  anemia.  It  does  not  seem  to  be  related  to  the 
adrenal.** 

LiPOCHROMES 

In  normal  plant  and  animal  tissues  occur  pigments  that  are  either 
fats  or  compounds  of  fat,  or  substances  highl}-  soluble  in  fats.  In 
animals  they  occur  normally  in  the  corpus  luteum,  in  the  epithelium 
of  the  seminal  vesicles,  testicles,  and  epididymis;  in  ganglion-cells, 
especially  in  the  sympathetic  nervous  tissue;  in  the  Kupffer  cells  of 
the  hver  and  in  fat  tissue.  Pathologically,  such  pigments  are  found 
particularly  in  the  muscle-cells  in  brown  atrophy  of  the  heart,  and 
less  abundantly  in  the  epithelium  of  atrophied  livers  and  kidneys 
(Lubarsch^^  and  Sehrt^")-  ^^1  are  characterized  b}^  staining  b}'  such 
fat  stains  as  sudan  III  and  scarlet  R,  and  usually,  but  not  constantly, 
by  osmic  acid;  they  are  dissolved  by  the  usual  fat  solvents.  It  is 
questionable  if  all  pigments  that  stain  for  fat  should  be  considered  as 
true  lipochromes,  however,  for  their  other  reactions  are  variable; 
and  Borst  would  distinguish  these  pathological  pigments  from  the 
true  lipochromes  by  calling  them  lipofuscins,  including  under  this  term 
the  brown  "waste  pigments, "  which  Hueck  believes  to  be  formed  from 
disintegrated  lipoids  or  fatty  acids.     Many  pigmentary  substances 

*i  See  Ingier,  Ziegler's  Beitr.,  1911  (51),  199. 

"  Frankfurter  Zeit.  Pathol.,  1913  (12),  218;  also  Teutschlaender,  Virchow's 
Arch.,  1914  (217),  393. 

••3  Jour.  Exp.  Med.,  1902  (6),  119. 

*Uour.  Exper.  Med.,  1911  (13),  290. 

"  Full  review  bv  McFarland,  Jour.  Amcr.  Med.  Assoc,  1917  (69),  1946. 

<»  Zcit.  phy-iol.  Chem.,  1913  (85),  92. 

"'  Mimch.  med.  Woch.,  1914  (61),  1332.  See  also  Hattori,  Mitt.  mod.  Ge- 
scUsch.,  Tokio,  191()  (30),  No.  6. 

"  See  Weber,  (>uart.  Jour.  Med.,  1919  (12),  404. 

"  Cent.  f.  Pathol.,  1902  (13),  881. 

*»  Virchow's  Arch.,  1904  (177),  248.  See  also  Mayer  ct  al,  Jour,  physiol.  et 
path.  g6n.,  1914  (16),  581. 


LIPOCIIROMES  479 

are  probably  soluble  in  fats,  and  in  this  way  t  lie  lipofusfins  are  formed.*' 
In  the  renal  epithelium  is  found  a  pigment  resembling  the  lipofuscins, 
increasing  with  age  and  not  related  to  the  urinary  pigments." 

Typical  plant  lipochromes,  as  also  the  pigments  of  Staphylococcus 
pyogenes  aureus  and  citreus,  are  colored  blue  by  concentrated  sulphuric 
acid  with  formation  of  small  blue  crystals  of  lipocyanin.  With  iodin- 
potassium-iodide  solution  they  are  colored  green.  Lipochrome  of 
frog-fat  stains  blue  with  this  solution  (Neumann);^'  lipochrome  of 
the  corpus  luteum  (called  lutein)  occasionally  gives  a  faint  blue  with 
sulphuric  acid  or  Lugol's  solution  (Sehrt);  but  the  fat-holding  pig- 
ments of  the  other  tissues  mentioned  above  do  not  give  either  of  these 
reactions.  Possibly  these  last  are  not  true  lipochromes,  therefore, 
but  rather  pigments  chemically  or  physically  combined  with  fat. 
Cotte''^  believes  that  the  true  lipochromes  of  plants  and  animals  have 
a  cholesterol  base,  but  the  presence  of  glycerol  in  plant  and  bacterial 
lipochromes  can  be  demonstrated  by  the  acrolein  test — possibly, 
therefore,  both  cholesterol  and  neutral  fats  are  present.  Melanins 
and  pigments  derived  from  hemoglobin  do  not  stain  with  sudan  III 
and  are  not  soluble  in  ether,  etc.,  and  hence  can  be  readily  distinguished 
from  the  fatty  pigments. 

It  has  been  shown  by  Escher*^  that  the  pigment  of  the  corpus  luteum 
is  identical  with  the  carotin  of  carrots.  Apparently  carotin  and  xan- 
thophyll  (a  crystalline  pigment  from  green  plants)  ^"^  are  the  chief 
pigments  of  milk  fats,  egg  yolk,  and  probably  of  body  fats.^^  In  the 
body  lipins  these  pigments  accumulate  throughout  life  because  of 
their  great  solubility  in  lipins,  which  explains  the  high  color  of  the  fats 
of  old  persons.  Carotin  seems  to  be  almost  or  quite  devoid  of  toxicity,  ^^ 
and  in  persons  eating  carrots  in  large  C|uantities  there  may  be  enough 
pigment  present  in  the  blood  {carotinemia)  to  produce  skin  pigmenta- 
tion resembling  jaundice.^" 

The  work  of  Palmer  indicates  that  carotin  and  xanthophyll  are 
much  more  widely  distributed  than  was  formerly  appreciated.  Ani- 
mals with  colored  fats  owe  the  color  to  these  plant  pigments,  which 
are  also  present  in  the  blood  of  these  same  animals,  but  not  in  the  blood 
of  animals  with  colorless  fats  (swine,  rabbits,  dogs,  sheep,  goats),  and 
the  so-called  lipofuscin  of  the  ganglion  cells  has  been  shown  to  be 

^^  Ciaccio  (Biochem.  Zeit.,  1915  (69),  313)  agrees  with  Hueck,  and  finds  it 
possible  to  distinguish  between  pigments  from  phosphatids,  which  stain  poorly 
with  Sudan  III,  and  those  from  free  fatty  acids  which  stain  deeply  with  this  dye. 

"  Schrever,  Frankf.  Zeit.  Pathol.,  1914  (15),  333. 

"  Virchow's  Arch.,  1902  (170),  363. 

"  Compt.  Rend.  Soc.  Biol.,  1903  (55),  812. 

"  Zeit.  physiol.  Chem.,  1913  (83),  198. 

"  Concerning  plant  pigments  see  review  by  West  and  Horowitz,Biochem.  Bullet., 
1915  (4),  151  and  161. 

^^  See  articles  by  Palmer  and  Eckles,  Jour.  Biol.  Chem.,  1914,  Vol.  17  et  seg. 

58  Wells  and  Hedenburg,  Jour.  Biol.  Chem.,  1916  (27),  213. 

"  Hess  and  Myers,  Jour.  Amer.  Med.  Assoc,  1919  (73),  1743;  see  also  ibid., 
1920  (74),  32. 


480  PATHOLOGICAL  PIGMENTATION 

carotin. '^^  Palmer  found  that  carotin  is  the  pigment  of  milk  fat, 
body  fat  and  corpus  luteum  of  the  cow,  while  xanthophyll  with  some 
carotin  colors  the  egg  yo\k,  body  fat  and  blood  serum  of  the  fowl. 
Chickens  deprived  of  these  pigments  from  the  time  of  hatching  have 
no  pigment  in  their  fats  or  egg  yolks  although  the  fowls  are  healthy 
and  their  colorless  eggs  are  fertile. ^^  This  work  makes  doubtful  the 
existence  of  other  fat-soluble  intracellular  pigments  in  man,  such  as 
lipofuscin,  and  Dolley  states  that  even  the  typical  lipofuscin  of  brown 
atrophy  of  the  heart  is  sometimes  insoluble  in  all  reagents  that  dissolve 
fats. 

Xanthosis  diabetica^^  also  seems  to  depend  on  an  excess  of  lipochromes  in  the 
blood,  probably  partly  endogenous  from  mobilization  of  tissue  fats  and  chiefly 
exogenous  from  the  abundance  of  green  vegetables  in  the  diet.  Accompanying 
hypercholesterolemia  is  usually  present. 

CMoroma." — The  pigment  that  causes  the  peculiar  green  color  characteristic 
of  these  malignant  growths,  was  considered  by  Chiari,  Huber  and  others  as  a 
fatty  substance  related  to  or  identical  with  the  lipochromes.  It  commonly  fades 
on  exposure  to  air,  and  also  when  in  the  usual  preservative  fluids,  to  which  it 
does  not  impart  its  color.  The  color  may  be  brought  back  after  formaldehyde 
preservation  by  H2O2  or  by  weak  alkalies  (Burgess)."  Ottenberg"  has  suggested 
that  the  green  color  may  be  due  to  eosinophiles  which  abound  in  chloromas,  since 
in  fresh  preparations  eosinophile  granules  have  a  faint  greenish  tinge.  It  contains 
no  iron,  is  soluble  in  absolute  alcohol  and  in  ether,  and  is  usually,  but  not  always 
(v.  Recklinghausen),  stained  black  with  osmic  acid.*^  Treadgold  states  that  as 
the  green  color  is  not  present  from  the  beginning  it  would  seem  that  cellular  de- 
generation must  play  a  part.  Possibly  a  degeneration  of  the  granules  of  the 
myelocytes  and  myeloblasts,  aided  by  the  products  of  hemoglobin  disintegration, 
is  responsible.*^^ 

Chromophile  cells  may  be  considered  in  this  connection.  Kohn^'  has  described 
certaincellswithadecidedaffinity  for  chromic  ocid  and  its  salts,  found  abundantly 
in  the  sympathetic  nervous  system,  in  the  carotid  gland,  and  in  the  medulla  of  the 
adrenal.  They  are  also  present  in  tumors  derived  from  these  organs.  Extracts 
from  such  organs  have  a  marked  effect  in  raising  blood  pressure,  and,  according 
to  Wiesel,'^*  they  are  greatly  involved  in  Addison's  disease.  The  nature  of  the 
chromophile  substance  is  unknown,  but  it  can  be  fixed  only  by  chromic  acid 
or  chromates;  cells  hardened  by  other  means  show  merelj'  spaces  in  the  places 
occupied  by  this  substance.  It  is  generally  believed  to  be  the  same  as  the  epi- 
nephrine, but  it  does  not  always  seem  to  parallel  in  amount  the  quantity  of  epine- 
phrine as  determined  chemically.  Ogata''"  states  that  the  chrome  reaction  depends 
on  the  reduction  of  chromic  acid  to  chromium  dioxide  by  epinephrine. 

«»  Dolley  and  Guthrie,  Jour.  Med.  Res.,  1919  (40),  295.  Marinesco,  however, 
says  that  the  pigment  of  nerve  cells  resembles  that  produced  during  autolysis  in 
ganglia  (C.  R.  Soc.  Biol.,  1913  (72),  838). 

6^  Jour.  Biol.  Chem.,  1919  (39),  299. 

62  Burger  and  Reinhart,  Ziet.  exp.  Med.,  1918  (7),  119. 

"Literature  by  Dock,  Amer.  Jour.  Med.  Sci.,  1893  (106),  152;  and  Dock  and 
Warthin,  Med.   News,  1904  (85),  971;  Burgess,  Jour.  Med.  Res.,  1912  (27),  133. 

«*  Amer.  Jour.  Med.  Sci.,  1909  (138),  505. 

*'  The  pigment  of  xanthelasma  multiplex  seems  to  be  a  fatty  substance  (Pocns- 
gen).     Virchow's  Arch.,  1883  (91),  354. 

«6  Quart.  .Jour.  Med.,  1908  (1),  239;  Weber,  Proc.  Roy.  Soc.  Med.,  Clin.  Med. 
Sec,  191G  (9),  7. 

"  Prag.  med.  Woch.,  1902  (27),  325. 

««  Zeit.  f.  Heilk.,  Path.  Alit.,  1903  (24),  257. 

«»  Jour.  Exp.  Med.,  1917  (25),  807. 


HEMOGLOBIN  481 

Blood  Pigments"" 

Red  corpuscles  behave  much  as  do  other  non-nucleated  fragments 
of  cells,  undergoing  disintegration  rapidly  and  constantly  when  under 
normal  conditions,  as  well  as  when  subjected  to  various  harmful  in- 
jfluences  (see  "Hemoh^sis"),  or  when  outside  of  the  vessels  in  extrava- 
sations of  blood.  The  processes  and  products  of  their  disintegration 
arc,  therefore,  much  the  same  whether  occurring  under  normal  or 
pathological  conditions.  The  hemoglobin  molecule  is  large  and  com- 
plex, and  from  it  are  derived  many  substances  of  the  nature  of  pig- 
ments; indeed,  hemoglobin  itself  may  appear  free  as  a  pigment. 

Hemoglobin  is  a  compound  protein,  consisting  of  a  protein  group 
{globin)  and  a  coloring-matter  {hematin  or  hemochromogen)  .''^  The 
protein  globin  is  of  a  basic  nature,  and  seems  allied  to  the  histons; 
the  hematin  is,  therefore,  presumably  acid.  Hemoglobin  ordinarily 
does  not  crystallize  readily,  especially  the  hemoglobin  of  man,  and  it 
is  doubtful  if  it  ever  does  so  in  the  living  tissues,  although  possibly 
this  may  occur  in  the  center  of  large  hematomas.  In  bodies  that  have 
undergone  postmortem  decomposition,  and  occasionally  in  specimens 
kept  for  microscopic  purposes,  irregular  orange-yellow  crystalline  masses 
of  hemoglobin  may  be  found.  This  occurs  particularly  if  the  blood 
has  been  acted  upon  by  hemolytic  agents  or  has  undergone  putrefactive 
changes,  and  then  is  hardened  in  alcohol.  The  crystals  are  either 
oxyhemoglobin,  or  more  often  an  isomeric  or  polymeric  modification, 
parahemoglohin  (Nencki).  Hemoglobin  also  enters  cells  unchanged, 
imparting  a  diffuse  yellowish  color,  and  apparently  it  is  non-toxic. ^^ 
If  present  in  the  blood  in  large  enough  amounts  it  is  excreted  un- 
changed in  the  urine,  but  at  least  one-sixtieth  of  the  total  number  of 
red  corpuscles  must  be  in  solution  at  one  time  to  produce  hemoglo- 
binuria; in  man  at  least  17  c.c.  of  laked  corpuscles  must  be  injected 
to  accomplish  this." 

Addis^'*  has  developed  the  following  conception  of  the  metabolism 
of  hemoglobin.  Free  hemoglobin,  liberated  especially  by  the  phago- 
cytes of  the  spleen,  is  taken  up  by  the  other  phagocytes,  notably  the 
Kupffer  cells  of  the  liver,  which  pass  it  on  to  the  liver  cells.  The 
pigment  moiety,  hematin  is  separated  from  the  globin,  and  converted 
through  removal  of  its  iron  into  bilirubin.  The  bilirubin  excreted  into 
the  intestine  is  there  reduced  to  urobilinogen,  which  is  in  part  reab- 
sorbed and  polymerized  into  urobilin,  which  in  turn  is  possibly  poly- 
merized into  a  larger  complex.  In  the  liver  this  urobilin  complex  has 
restored  to  its  pyrrol  nuclei  the  original  side  chains,  and  then  is  used 

^"Literature  by  Schmidt,  Ergebnisse  der  Pathol.,  1894  (!■>),  101;  and  1896 
(nil),  542;  Schulz,  Ergebnisse  der  Physiol.,  1902  (Ii),  505. 

'^  Halliburton  and  Rosenheim  recommend  that  the  name  "hemochromogen" 
be  dropped  in  favor  of  "reduced  hematin'.'  (Biochem.  Jour.,  1919  (13),  195). 

72  Barratt  and  Yorke,  Brit.  Med.  Jour.,  Jan.  31,  1914. 

"  Sellards  andMinot,  Jour.  Med.  Res.,  1916  (34),  469. 

'*Arch.  int.  Med.,  1915  (15),  412. 

31 


482  PATHOLOGICAL  PIGMENTATION 

to  form  new  hemoglobin  molecules.  This  hj^pothesis  is  merely  ten- 
tative, but  it  affords  a  useful  "working  hypothesis"  for  the  considera- 
tion of  many  phases  of  pigment  metabolism. 

In  the  decomposition  of  hemoglobin  the  first  step  is  the  splitting 
of  the  globin  (which  does  not  form  pigments)  from  the  hematin,  from 
which  many  pigments  may  be  derived. 

Hematin. — The  formula  given  for  this  substance  by  Nencki  is 
C32H32N4Fe04  while  Hoppe-Seyler  proposed  the  formula  C34H34 
N4Fe05,  although  it  is  not  certain  that  the  hematin  of  all  animals  is 
the  same.  It  is  found  frequently  as  an  amorphous,  dark-brown  or 
bluish-black  substance,  in  large,  old  extravasations  of  blood,  but  sel- 
dom in  small  hemorrhages.  As  a  pathological  pigment  hematin  is 
by  no  means  so  frequently  found  as  its  derivatives.  Schumm^^  ob- 
served a  patient  with  chromium  poisoning  w^ho  showed  for  several 
days  abundant  hematin  free  in  the  blood.  He  has  also  found  it  in 
malaria,  pernicious  anemia,  congenital  hematoporphj^ria,  and  gener- 
ally with  acute  toxic  hemolysis,  including  patients  infected  with  B. 
eniphysematosus,  when  the  hematin  may  be  accompanied  by  me+he- 
moglobin  without  a  corresponding  urinary  excretion  of  these  pigments. 
Feigl  found  hematinemia  in  many  cases  of  poisoning  with  the  war  gases.  "^ 
Brown"  found  that  solutions  of  hematin  cause  chills  and  fever,  and 
suggests  that  his  pigment  may  be  at  least  partially  responsible  for 
the  symptoms  of  malaria. '^^  Hematin  has  been  beheved  to  spht  up 
gradually  into  an  iron-free  pigment  {hematoidin)  and  an  iron-contain- 
ing pigment  {hemosiderin).  This  change  may  be  represented  by  the 
following  equation,  according  to  Nencki  and  Sieber:^^ 

C32H32N404Fe  +  2H2O  =  2C,6H,8N203  +  Fe. 
(hematin)  (hematoidin) 

However,  finding  that  the  pigment  in  the  malarial  spleen  is  hematin, 
Brown^"  suggests  that  hematin  cannot  well  be  an  intermediary  prod- 
uct in  hemoglobin  disintegration,  since  this  malarial  pigment  persists 
a  very  long  time  in  the  tissues  without  change.  He  has  made  other 
observations  that  led  him  to  conclude  that  hematin  is  not  an  inter- 
mediary substance  between  hemoglobin  and  hemosiderin,  but  that 
when  once  formed  it  is  destroyed  very  slowly,  by  oxidation  rather  than 
hydrolysis.  Injected  into  rabbits  it  produces  vascular  lesions  in  the 
kidneys**'  and  in  large  doses  causes  a  marked  fall  in  blood  pressure. *- 
Hematoidin  may  be  found  in  old,  large  extravasations,  as  orange- 

"  Zeit.  physiol.  Chem.,  1912  (80),  1;  1913  (87),  171;  1916  (97),  32. 
'«  Biochem.  Zeit.,  1919  (93),  119. 
"  Jour.  Exper.  Med.,  1912  (15),  580;  1913  (18),  96. 

'8  Disputed  by  Butterfield  and  Benedict,  Proc.  Soc.  Exp.  Biol.,  1914  (11),''80. 
"Arch.  exp.  Path.  u.  Pharm.,  1888  (24),  440;  Brugsch  and  Vo.slumoto,  Zeit. 
exp.  Path.,  1911  (8),  639. 

"ojour.  Exper.  Med.,  1911  (13),  290;  1911  (14),  612. 

«'  Arch.  Int.  Med.,  1913  (12),  315. 

"  Brown  and  Loevenhart,  Jour.  Exp.  Med.,  1913  (IS),  107. 


BLOOD  PIGMENTS  483 

colored  or  red  rhombic  plates,  first  described  by  Virchow.  Some- 
times, however,  hematoidin  occurs  in  the  form  of  yellowish  granular 
masses,  and  it  may  be  associated  with  lipoids;  it  is  also  found  irt 
crystalline  form  in  icterus  (Dunzelt).^'  It  seems  to  be  nearly  or 
quite  identical  with  the  bile-pigment,  bilirubin,  and  it  is  probably  the 
source  of  this  substance  under  normal  conditions.  When  formed  in 
excessive  amounts,  either  through  increased  destruction  of  corpuscles 
in  the  vessels  or  in  extravasations,  the  amount  of  bile-pigment  is  in- 
creased (see  "Icterus").  Possibly  some  of  the  hematoidin  becomes 
transformed  (Urectly  into  urobilin,  and  is  then  eliminated  in  the  urine. 

Hemosiderin'*''  is  relatively  insoluble,  and,  therefore,  is  more 
slowly  removed  when  formed  in  hemorrhages,  and  more  abundantly 
deposited  in  the  tissues  when  formed  after  excessive  hemolysis.  In 
acute  hemolytic  anemia  a  third  of  the  total  iron  of  the  blood  may  be 
deposited  in  the  liver,  spleen  and  kidneys  within  24  hours. ^'^  In 
infarcts  hemosiderin  soon  disappears  (Schmidt), ^"^  presumably  because 
dissolved  by  the  acids  formed  during  autolysis.  According  to  Neu- 
mann, hemosiderin  is  produced  only  under  the  influence  of  living  cells 
and  in  the  presence  of  oxygen,  while  hematoidin  arises  independent 
of  cellular  activity;^^  but  Brown^^  has  found  that  hemosiderin  can  be 
formed  during  autolysis  of  the  liver,  especially  when  air  is  present, 
and  therefore  probably  by  an  oxidizing  enzyme.  He  suggests  that  in 
hemosiderin  the  pigment  is  still  hematoidin,  and  that  the  formation 
of^hemosiderin  takes  place  in  the  nuclei,  the  hemosiderin  being  made 
directly  from  hemoglobin  without  the  intervention  of  hematin.  It 
may  also  be  formed  from  the  iron-containing  protein  of  the  cells  during 
autolysis,  independent  of  hemoglobin. ^^  Milner^"  considers  that, 
under  similar  conditions,  an  iron-containing  pigment  is  also  formed, 
which  differs  from  hemosiderin  in  having  the  iron  so  combined  that 
it  cannot  react  with  the  usual  reagents;  this  pigment  may  later  change 
into  hemosiderin.  Up  to  the  present  time  we  do  not  know  the  chemi- 
cal nature  of  hemosiderin,  nor  its  exact  fate  in  the  body,  but  it  is 
probably  utilized  in  the  manufacture  of  new  hemoglobin,  for  it  is 
known  that  the  iron  liberated  when  hematin  is  broken  up  in  the  body 
under  experimental  conditions  is  deposited  and  not  eliminated  (Mor- 
ishima).^^ 

Unstained  hemosiderin  generally  appears  in  the  form  of  brown 

*^  Cent.  f.  Path.    1909  (20)   966. 

8-«  See  Neumann!  Virchow's  Arch.,  1888  (111),  25;  1900  (161),  422;  1904  (177), 
401;  also  Arnold,  ibid.,  1900  (161),  284;  Leupold,  Beitr.  path.  Anat.,  1914  (59),  501. 

s^Muir  and  Dunn,  .Jour.  Path,  and  Bact.,  1915  (19),  417. 

86  Verh.  Deut.  Path.  Gesell.,  1908  (12),  271. 

8^  The  accumulation  of  iron  in  the  liver  which  follows  poisoning  with  hemolytic 
agents,  is  not  prevented  or  diminished  bv  preliminary  removal  of  the  spleen 
(Meinertz,  Zeit.  exp.  Path.  u.  Ther.,  1906  (2),  602). 

S8  Jour.  Exper.  Med.,  1910  (12),  623. 

83  Sprunt  et  al,  Jour.  Exp.  Med.,  1912  (16),  607. 

90  Virchow's  Arch.,  1903  (174),  475. 

»i  Arch.  exp.  Path.  u.  Pharm.,  1898  (41),  291. 


484  PATHOLOGICAL  PIGMENTATION^ 

or  yellowish-brown  granules,  and  not  as  crystals.  After  a  time  it  is 
taken  up  and  deposited  to  a  large  extent  in  the  liver,  spleen,  bone- 
jnarrow,  and  kidney,  either  as  hemosiderin  or  possibly  as  some  other 
iron  compound  of  similar  nature.  From  these  sites  it  seems  to  be 
later  taken  up  to  be  utilized  in  the  manufacture  of  new  red  corpuscles. 
Whenever  there  is  hemosiderin  deposition  in  the  kidney,  granules 
of, the  pigment  may  be  found  in  the  urine,  free  or  in  cells  (Rous).^^ 

All  told  the  average  human  body  contains  about  3.2  grams  of  iron, 
of  which  2.4  to  2.7  grams  is  in  the  blood.  According  to  ]\Ieyer^^ 
iron  is  present  in  the  body  in  three  forms:  1.  Not  demonstrable  by 
reagents  because  so  firmly  bound  (hemoglobin).  2.  Loosely  bound 
iron,  colored  by  (NHJaS  acting  for  a  long  time  (ferratin).  3.  Salt- 
like compounds  with  proteins,  and  inorganic  compounds,  reacting  at 
once  with  reagents.  Ferratin  is  the  iron  compound  in  the  liver,  con- 
taining 6  per  cent.  iron.  He  believes  that  probably  hemosiderin  is 
not  a  definite  substance,  but  merely  indicates  compounds  of  the  third 
class.  Iron  pigments  may  be  transformed  from  one  class  to  another, 
e.  g.,  in  corpus  luteum  scars,  whose  age  can  be  estimated,  class  three 
may  be  replaced  by  class  two.  We  may  have  in  the  sputum  and  lungs 
"  Herzf ehlerzellen "  that  either  do  or  do  not  stain  with  ferrocyanide. 
In  morbus  maculosus,  Kunkel  found  the  pigment  of  the  internal  organs 
to  be  pure  iron  oxide.  Hueck  also  holds  that  hemosiderin  is  an  in- 
organic iron  compound,  loosely  bound  to  proteins  and  fats,  and  that 
it  never  forms  an  iron-free  pigment,  as  has  been  stated.  He  believes 
that  there  is  very  little  iron  in  the  tissues  in  a  firm  union  like  hemo- 
globin, and  that  by  proper  technic  some  iron  can  be  stained  in  every 
organ  which  contains  iron  chemically  demonstrable.  Ischida''*  be- 
lieves that  an  iron-containing  pigment  may  be  formed  in  striated 
muscles  from  the  iron  normally  there,  without  requiring  a  hematoge- 
nous origin. 

Hematoporphyrin.^^ — -There  are  several  closely  related  pigments  de- 
rived from  hematin  that  are  appropriately  grouped  under  the  desig- 
nation of  porphyrins,  for  they  are  not  all  identical  with  the  pigments 
prepared  artificially  from  hematin  by  Nenclci  and  called  by  him  hema- 
topoi'phyrin  and  mesoporphyrin ,  the  former  apparently  representing  a 
reduction,  the  latter  an  oxidation  product. "''  The  porphj-rins  found 
in  the  urine  and  feces  are  different  from  each  other  and  from  those 
prepared  by  Nencki."  Physiologically,  these  pigments  are  of  great 
interest,  because  of  the  close  chemical  relation  they  have  been  found 

"  Jour.  Exp.  Med.,  1918  (28),  645. 

"  Ergel).  der  Physiol.,  190.5  (5),  698;  literature. 

»^  Virchow's  Arch.,  1912  (210),  67. 

*'  Literature  and  full  review  by  Giinther,  Deut.  Arch.  klin.  I\Icd.,  1912  (105), 
89;  and  by  Jcsionek,  Ergeb.  inn.  Med.,  1913  (II),  525. 

»•  Fischer  and  Meyer-Betz,  Zeit.  physiol.  Chem.,  1912  (82),  96. 

•7  H.  Fischer,  Munch,  med.  Woch.,  1916  (63),  377;  Zeit.  physiol.  Chem.,  1916 
(97),  109  and  148;  Schuinin,  ibid.,  1915  (96),  183. 


BLOOD  PIGMENTS  485 

to  bear  to  chlorophyll,^'^  with  wliicli  luMiioslobin  is  so  closely  rolatod 
functionally.  It  is  also  interesting  to  consider  that  whereas  carnivora 
obtain  much  hemoglobin  in  their  food,  herbivora  obtain  much  chlo- 
rophyll. Pathologically,  porphjTin  is  of  interest  as  a  urinary  pigment, 
being  found  normally  in  the  urine  in  traces,  but  present  in  considerable 
quantities  in  many  diseases, ^^  such  as  rheumatism,  tuberculosis, 
various  liver  diseases,  and,  most  strikingly,  after  the  administration 
of  sulphonal,  veronal  or  trional.  A  congenital  form  of  hematoporphy- 
ria  occurs,  in  which  the  blood  contains  free  hematin  and  a  porphyrin 
(Schumm),^  about  0.3-0.4  gm.  being  usually  excreted  daily  in  the  urine; 
in  the  blood  it  is  accompanied  by  hematin  and  bilirubin.  When  in  abun- 
dance it  may  color  the  urine  a  rich  Burgundy  red,  and  it  is  sometimes 
accompanied  by  a  precursor,  uro-fuscin.  It  is  present  in  the  bones  of 
animals  showing  hemochromatosis  and  in  the  bones  of  persons- 
exhibiting  the  congenital  form  of  "hematoporphyria,"  described  by 
Giinther,  which  is  accompanied  by  severe  skin  lesions  that  are  ascribed 
to  the  action  of  light  upon  the  skin  sensitized  by  the  hematoporphyrin. 
Hausmann''  and  others  have  studied  extensively  the  photosensitizing 
action  exhibited  by  hematoporphyrin  and  other  porphyrins,  and  find 
evidence  suggesting  a  relationship  between  hematoporphyria  and 
"  hydroa  aestiva,"  and  other  conditions  in  which  the  skin  is  abnormally 
sensitive  to  light.  An  acute  form  of  porphyrinuria  has  been  described, 
usually  in  women,  and  sometimes  associated  with  ascending  motor 
paralysis.''" 

Afterinjection  of  0.2  gm.  hematoporphyrin  into  his  own  veins  Meyer- 
Betz'*  found  himself  so  sensitized  to  light  that  exposure  to  the  sun 
caused  severe  skin  reactions  during  a  period  of  weeks,  and  exposure 
to  the  Finsen  light  produced  severe  ulceration;  but  little  hemato- 
porphyrin escaped  in  the  urine.  Many  other  products  of  blood 
destruction  tested  on  animals  were  without  sensitizing  effects.  IVIeth- 
ylation  of  the  pyrrol  groups  only  partially  removes  the  activity  of 
hematoporphyrin.  Porphyrin  obtained  from  urine  and  feces  by  Fischer 
also  sensitized  mice  to  light.  Sufficient  doses  of  hematoporphyrin 
may  sensitize  mice  so  that  they  become  narcotized  and  die  in  a  few 
minutes  after  exposure  to  intense  light,  a  true  "light  stroke." 

Pseudomelanosis. — When  loosely  bound  iron  is  present  in  the 
tissues,  and  in  the  same  tissues  sulphides  are  produced  tlirough  bac- 
terial action,  a  discoloration  with  sulphide  of  iron  will  result,  which  is 
called  pseudomelanosis,  because  the  pigment  resembles  true  melanin 
in  its  blackness.     This  is  most  frequenth'-  observed  as  a  postmortem 

'^  For  literature  see  Abderhalden,  "Lehrbuch  der  physiol.  Chemie,"  1906. 
95  See  Garrod,  .Jour,  of  Physiol.,  1892  (13),  598. 

iZeit.  physiol.  Chem.,  1916  (98),  123;  1919  (105),  158. 

'  Hegler  et  al,  Deut.  med.  Woch.,  1913  (39),  842. 

=  Biochein.  Zeit.,  1910  (30),  276;  1914  (67).  309. 

3<»L6ttier,  Corr.-bl.  f.  Schweizer  Aerzte,  1919  (49),  1871. 

*  Deut.  Arch.  klin.  Med.,  1913  (112),  476. 


486  PATHOLOGICAL  PIGMENTATION 

phenomenon  in  and  about  the  abdominal  cavity,  and  in  the  ordinary 
postmortem  discoloration  both  the  liberation  of  the  iron  from  its 
firm  organic  combination,  and  the  production  of  hydrogen  sulphide, 
are  the  work  of  bacteria.  Pseudomelanosis  may  occur  intra  vitam, 
particularly  in  the  margins  of  infected  areas,  and  it  may  also  be  ob- 
served in  the  intestines,  liver  and  spleen,  and  about  the  peritoneum, 
in  bodies  examined  immediately  after  death,  before  any  evident  post- 
mortem decomposition  has  set  in.  This  seems  to  depend  upon  the 
previous  intra  vitam  formation  of  hemosiderin,  which  is  then  combined 
by  sulphur  liberated  from  tissue  proteins  through  bacterial  action.^ 

Methemoglobin. — If  hydrogen  sulphide  acts  upon  hemoglobin 
that  has  not  been  decomposed,  a  greenish  compound  of  sidphur- 
methemoglobin  is  formed  (Harnack^),  which  is  the  cause  of  the  greenish 
color  seen  in  the  abdominal  walls  and  along  the  vessels  of  cadavers. 
This  union  of  hemoglobin  and  hydrogen  sulphide  occurs  only  when 
oxygen  is  present  (oxyhemoglobin).  The  sulphur-hemoglobin  com- 
pound is  readily  decomposed  by  weak  acids,  even  by  CO2,  with  the 
formation  of  methemoglobin,  which  in  turn  readily  becomes  decomposed 
to  form  hematin.  During  life  sulphemoglobin  may  form  in  the  cir- 
culating blood,  the  sulphur  presumably  coming  from  intestinal  putre- 
faction, and  hence  the  condition  is  called  "enterogenous  cyanosis," 
which  term  also  covers  methemoglobinemia  produced  by  nitrites  formed 
in  the  intestines. '^  The  latter  condition  is  also  present  in  poisoning 
by  phenacetin,^  aniline  and  acetanilid,  and  related  pigments  appear 
in  the  blood  in  poisoning  with  chlorates  and  nitrobenzol.  Pneu- 
mococci  and  Streptococcus  viridans,  as  well  as  some  other  bacteria, 
may  produce  methemoglobin.^  In  infections  with  B.  em-physem.atosus , 
Schumm  found  this  pigment  free  in  the  blood.  Van  den  Bergh^° 
has  found  sulphemoglobinemia  in  puerperal  sepsis,  and  probably  these 
pigments  could  be  found  in  other  conditions  if  sought. 

Hemof  uscin  is  the  name  given  by  von  Recklinghausen  to  the 
brownish  pigment  found  in  involuntary  muscle-fibers,  particularly  in 
the  wall  of  the  intestine.  It  does  not  react  for  iron,  and  is  insoluble 
in  alcohol,  ether,  chloroform,  or  acids;  therefore  it  is  not  a  lipochrome. 
It  is  bleached  by  H2O2,  and  is  often  found  associated  with  hemosiderin 
which  is  not  bleached.  Von  Recklinghausen,  and  also  Goebel,^' 
ascribe  this  pigment  to  an  alteration  of  hemoglobin  which  enters  the 
cells  in  dissolved  form,  but  Rosenfeld,^-  who  has  subnutted  the  mater- 

'  Ernst,  Virchow's  Arch.,  1898  (152),  418.     Literature. 

«  Zeit.  physiol.  Chem.,  1899  (2G),  558. 

'  West  and  Clarke,  Lancet,  Feb.  2,  1907;  Davis,  ibid.,  Oct.  26,  1912;  Gibson 
Quart.  Jour.Med.,  1907  (1),  29;  Long  and  Spriggs,  /6/(/.,  1918  (IH,  102;  .Tamieson, 
ibid.,  1919  (12),  81. 

8  See  Heuhner,  Arch.  exp.  Path.,  1913  (72),  241. 

9  Cole,  Jour.  Kxp.  Med.,  1914  (20),  303;  Blake  ibid.,  1916  (24),  315;  Schumm, 
Zeit.  physiol.  (Miem.,  1913  (87),  171. 

"•  Nederl.  Tijd.  Geneesk.,  1918  (1),  1774. 

"  Virchow's  Arch.,  1894  (136),  482. 

"  Arch.  exj).  Path.  u.  Pharin.,  1900  (45),  46. 


HEMOCIIROMA  TOSI S  487 

iai  to  analysis  after  isolation,  found  3.70  per  cent,  of  sulphur,  from 
which  he  considers  that  it  is  related  to  the  nielanins  or  melanoid  sub- 
stances. The  substance  is  readily  dissolved  by  alkalies,  and  con- 
tains no  iron.  According  to  Taranoukhinc,'''  the  pigment  in  the  myo- 
cardium in  brown  atrophy  of  the  heart  is  also  derived  from  proteins,  and 
is  neither  a  lipochrome  nor  a  hemoglobin  derivative.  Other  observers, 
however,  consider  this  pigment  a  lipochrome  or  a  lipofuscin.  It  is 
probable  that  the  name  hemofuscin  has  been  given  to  several  different 
pigments,  which  resemble  one  another  only  in  that  they  do  not  con- 
tain iron.  Strater^"*  says  that  the  name  hemofuscin  cannot  be  used  for 
the  pigment  of  the  involuntary  muscles,  as  he  finds  evidence  that  it 
does  not  arise  from  hemoglobin  and  is  probably  a  waste  pigment;  but 
hemofuscin  is  found  in  epithelial  and  connective  tissue  cells. 

Hemochromatosis."'' — -This  name  was  given  by  von  Reckling- 
hausen to  a  condition  in  which  the  organs  and  tissues  throughout  the 
body  are  abundantly  infiltrated  with  two  pigments;  one,  iron-con- 
taining, identical  with  hemosiderin;  the  other  seems  to  be  the  same 
as  the  hemofuscin  described  above.  It  is  usually  distinguished  from 
general  hemosiderosis  in  which  only  the  iron  pigment  is  deposited, ^^ 
although  there  are  numerous  observers  who  believe  that  all  the  pigment 
in  hemochromatosis  contains  iron,  but  in  some  of  the  pigment  the 
iron  is  firmly  bound  and  difficult  of  demonstration.  The  hemosiderin 
is  found  chiefly  in  the  parenchyma  cells  of  the  glandular  organs,  espec- 
ially the  liver  and  pancreas,  which  organs  usually  show  marked  inter- 
stitial proliferation.  The  hemofuscin  is  found  in  the  smooth  muscle 
fibers  of  the  gastro-intestinal  tract,  blood  vessels,  and  genito-urinary 
tract.  Under  the  heading  of  local  hemochromatosis,  von  Reckling- 
hausen grouped  such  conditions  as  brown  atrophy  of  the  heart  and 
pigmentation  of  the  intestinal  wall,  which  probably  are  quite  distinct 
from  the  generalized  hemochromatosis,  since  the  local  form  occurs  as  a 
physiological  process  in  old  age.  Hess  and  Zurhelle  found  38.7  gm. 
of  iron  in  the  liver  in  one  case  (the  normal  amount  is  0.3  gm.),  and 
Bernouille'^^  found  18.3  gm.  or  2.95  per  cent,  of  the  dry  weight  in 
the  liver,  2.65  per  cent,  in  the  pancreas,  and  the  same  in  the  spleen. 
Anschiitz  found  14.69  per  cent,  in  the  lymph  glands,  7.62  per  cent,  in 
the  liver,  and  5  per  cent,  in  the  pancreas  of  a  case.  Muir  and  Dunn^^ 
obtained  the  following  percentage  figures:  Liver,  6.43;  pancreas, 
2.49;  spleen,    0.825;  retroperitoneal    glands,    11.64;  kidneys,    0.406; 

13  RousskyArch.  Patol.,  1900  (10),  441.      ,. 

1^  Virchow's  Arch.,  1914  (218),  1. 

15  Literature  given  by  Sprunt,  Arch.  Int.  Med.,  1911  (81,  75;  Potter  and  Milne, 
Amer.  Jour.  Med.  Sci.,  1911  (143),  46;  Roth,  Deut.  Arch.  klin.  Med.,  1915  (117), 
224;  McCreery,  Canada  Med.  Assoc.  Jour.,  1917  (7),  481;  Howard  and  Stevens, 
Arch.  Int.  Med.,  1917  (20),  896. 

'^  In  lower  animals  occurs  a  form  of  hemochromatosis  affecting  especially  the 
bones,  and  sometimes  mistaken  for  ochronosis.  (See  Teutschlacnder,  \'irchow's 
Arch.,  1914  (217),  393.) 

1'  Corr.-Bl.  Schweiz.  Aertze,  1911  (40),  610. 

18  Jour.  Path,  and  Bact.,  1914  (19),  226. 


488  PATHOLOGICAL  PIGMENTATION 

adrenals,  0.121;  heart,  0.714;  skin,  0.188;  small  intestine,  0.14.  (Other 
analytical  results  are  given  by  Howard  and  Stevens.) 

Opie's  conclusions  concerning  this  disease  are  as  follows:  (1) 
There  is  a  distinct  morbid  entity,  hemochromatosis,  characterized 
by  widespread  deposition  of  an  iron-containing  pigment  in  certain 
cells,  and  an  associated  formation  of  iron-free  pigments  in  a  variety 
of  locahties  in  which  pigment  is  found  in  moderate  amount  under 
physiological  conditions.  (2)  With  the  pigment  accumulation  there 
occur  degeneration  and  death  of  the  containing  cells  and  consequent 
interstitial  inflammation,  notably  of  the  liver  and  pancreas,  which 
become  the  seat  of  inflammatory  changes  accompanied  by  hyper- 
trophy of  the  organ.  (3)  When  chronic  interstitial  pancreatitis 
has  reached  a  certain  grade  of  intensity,  diabetes  ensues  and  is  the 
terminal  event  in  the  disease. 

Diabetes  occurs  in  the  majority  of  the  cases  of  generalized 
hemochromatosis  (50  of  63  collected  by  Sprunt)  and  was  called  by 
Hanot,  "bronzed  diabetes,"  because  of  the  coloration  of  the  skin. 
It  has  been  suggested  that  the  pigmentation  is  due  to  decomposition 
of  the  blood-corpuscles  in  the  diabetic  blood,  but  the  pigmentation 
and  sclerotic  changes  precede  the  diabetes,  which  is  secondary  to  the 
atrophic  and  sclerotic  changes  in  the  pancreas.  It  seems  probable  that 
both  the  pigment  formation  and  the  tissue  changes  depend  upon  some 
intoxication,  the  origin  and  nature  of  the  toxic  agent  being  entirely 
unknown.  In  many  cases  it  has  seemed  possible  that  alcohol  might 
have  been  the  inciting  cause.  There  is  no  evidence  of  abnormal  boold 
destruction  which  might  account  for  the  pigmentation,  and  ]\leltzer 
and  Parker  have  suggested  that  the  difficulty  lies  in  the  inability  of 
the  tissues  to  get  rid  of  the  iron  set  free  in  normal  catabolism.  INIetab- 
olism  studies  have  indicated  that  there  is  some  retention  of  food  iron 
which  may  be  interpreted  as  supporting  but  not  proving  this  hypothe- 
sis.^^ Rous  and  Oliver, -°  finding  that  protracted  hemolysis  of  foreign 
corpuscles  in  rabbits  produces  a  typical  mild  hemochromatosis,  sug- 
gest that  the  liver  cirrhosis  is  primary  and  renders  this  organ  unable 
to  deal  adequately  with  the  blood  pigments,  which  therefore  accumu- 
late in  the  organs  and  cause  diffuse  fibrosis. 

ICTERUS^' 

Pigmentation  of  the  tissues  of  tlie  body  in  jaundice  depends  upon 
the  presence  in  them  of  bile-pigments,  which  usually  have  been  formed 
in  the  liver  and  reabsorbed  either  into  the  lymph  or  blood  (or  botii). 
However,  a  pigment  that  seems  to  be  chemically  identical  with  bili- 
rubin (hematoidin)  may  be  formed  from  hemoglobin  liberated  on  the 

"  See  Howard  and  Stevens  {loc.  cit.)  and  McCluro,  Arch.  Int.  Med.,  1918  (22), 
610. 

2"  Trans.  Assoc.  Anier.  Phys.,  1918  (3.3;,  132. 

2' Literature  l)y  Stadelinann,  "Der  Icterus,"  Stuttgart,  1891;  Minkowski, 
Ergebnisse  der  Pathol.,  1S9.')  (2),  079. 


ICTERUS  489 

breaking  up  of  red  corpuscles,  and  possibly  this  may  be  produced  in 
sufficient  amounts  outside  of  the  liver  to  give  rise  to  general  icterus. 
Certainly  the  local  greenish-yellow  pigmentation  occurring  in  the 
vicinity  of  extravasations  of  blood,  due  to  hematoidin  formation,  may 
be  looked  upon  as  a  "local  jaundice,"  and  in  icterus  hematoidin^^ 
crystals  may  be  found  in  the  tissues.-^ 

Bile-pigments. — Bilirubin  is  of  a  reddish-yellow  color,  and  it  Is  the  chief  pig- 
ment of  human  bile.  Its  formula  is  CsiHssN-iOe  or  CssHjeN^Oe,  and  its  relation  to 
hematin,  from  which  it  is  formed,  is  shown  by  the  following  formula,  which  ex- 
presses the  manner  in  which  blood  pigment  may  be  converted  into  bilirubin  by 
the  liver  under  normal  conditions,  and  into  hematoidin  (its  isomer)  in  the  tissues 
and  fluids  of  the  body  in  pathological  conditions: 

CaiHa^N.OjFe  +  2H2O  =     C3.H38N4O6     +  FeO. 
(hematin)  (hematoidin  or 

bilirubin) 

Bilirubin  is  not  soluble  in  water,  but  dissolves  in  the  alkaline  body  fluids  as  a 
soluble  compound,  "bilirubin  alkali."  It  is  very  slightly  soluble  in  ether,  ben- 
zene, carbon  disulphide,  amyl-alcohol,  fatty  oils,  and  glycerol,  but  is  more  soluble 
in  alcohol  and  in  chloroform. 

Biliverdin,  0^411:1^^40$,  as  its  formula  indicates,  is  an  oxidation  product  of 
bilirubin.  Bilirubin  in  alkaline  solutions  will  oxidize  into  biliverdin  merely  on 
exposure  to  the  air,  and  the  change  from  yellow  to  green  of  icteric  specimens  when 
placed  in  oxidizing  solutions  (e.  g.,  dichromate  hardening  fluids)  is  due  to  the 
formation  of  the  green  biliverdin.  Biliverdin  is  the  chief  pigment  of  the  bile  of 
carnivora,  but  it  is  also  present  in  varying  amounts  in  human  bile. 

The  various  other  biliary  pigments,  namely,  bilifuscin,  biliprasin,  choleprasin,-* 
bilihumin,  and  bilicyanin,  are  probably  not  normal  constituents  of  bUe,  but  are 
oxidation  products  of  bilirubin,  and  are  found  chiefly  in  gall-stones  (q.  v.).  A 
pigment  similar  to  urobilin  maj'-  be  present  in  normal  bile.  The  total  amount  of 
pigments  present  in  bile  is  probably  not  far  from  one  gram  per  liter;  rather  under 
than  above  this  amount. 

Etiology  of  Icterus. — ^Although  hematoidin,  which  is  isomeric  if 
not  identical  with  bilirubin,  may  be  formed  outside  of  the  liver  when 
red  corpuscles  are  broken  up  in  hemorrhagic  extravasations,  and 
possibly  also  when  they  are  broken  up  within  the  vessels  by  hemolytic 
agents,  yet  it  was  formerly  held  that  a  true  general  icterus  does  not 
occur  without  the  liver  being  implicated.  This  view  rested  on  evi- 
dence of  various  sorts.  First,  the  classical  experiments  of  Minkowski 
and  Naunyn,-^  which  demonstrated  that  in  geese  the  production  of 
hemolysis  by  means  of  arseniuretted  hydrogen  leads  to  icterus,  but  if 
the  livers  of  the  geese  have  been  previously  removed,  no  icterus  follows 
the  poisoning.  Second,  the  repeated  demonstration  that  in  icterus 
produced  by  septic  conditions,  poisoning,  etc.,  which  was  formerly 
looked  upon  as  Si  "hematogenous"  icterus,  the  urine  contains  bile 
salts  as  well  as  pigment,  indicating  an  absorption  of  bile  from  the  liver. 
Third,  the  finding  of  histological  evidence  that  in  so-called  hematogen- 

"  See  Guillain  and  Troisier,  Semaine  Med.,  1909  (29),  133;  Widal  and  Joltrain, 
Arch.  med.  expcr.,  1909  (21),  641. 

•"  Dunzelt,  Cent.  f.  Path.,  1909  (20),  966. 

24  SeeKiister,  Zeit.  physiol.  Chem.,  1906  (47),  294. 

"  Arch.  f.  exp.  Pathol,  u.  Pharm.,  18S6  (21),  1. 


490  PATHOLOGICAL  PIGMENTATION 

ous  icterus  there  occur  occlusions  or  lesions  of  some  sort  in  the  bile 
capillaries,  which  can  account  for  the  reabsorption  of  the  bile  into  the 
general  circulation. ^^^  Therefore,  it  was  beheved  that  the  pigments 
that  produce  the  general  discoloration  of  icterus  are,  at  least  for  the 
most  part,  manufactured  by  the  liver,  whatever  the  cause  of  the  re- 
absorption  of  the  bile  from  the  Hver  into  the  blood  may  be.  That 
hemolytic  agents  cause  icterus  was  explained  by  the  fact  that  on 
account  of  the  large  amounts  of  free  hemoglobin  brought  to  the  liver, 
excessive  amounts  of  bile-pigments  are  formed,  which  render  the  bile 
so  viscid  that  it  blocks  up  the  fine  bile  capillaries;  on  account  of  the  low 
pressure  at  which  bile  is  secreted,  a  slight  obstruction  of  this  kind  is 
sufficient  to  stop  entirely  the  outflow  of  bile,  which  then  enters  the 
capillaries  of  the  liver  and  also,  to  a  less  extent,  the  lymphatics.-^  It 
is  also  possible  that  the  hemolytic  poisons  injure  the  liver-cells  so 
much  that  the  minute  intra-  and  intercellular  bile  capillaries  become 
disorganized,  and  permit  of  escape  of  bile  into  the  lymph-spaces  and 
its  absorption  into  the  blood-vessels.  ^^  Swelling  of  the  degenerated 
liver-cells  may  also  be  an  important  factor  in  the  occlusion  of  the  bile 
capillaries;  swelHng  of  the  lining  cells  of  the  bile  capillaries  may  also 
coexist,  and  fibrin  may  occlude  them  in  toxic  or  infectious  icterus. 

However,  Whipple  and  Hooper-^  have  obtained  experimental  evi- 
dence that  after  intravenous  injection  of  hemoglobin  into  dogs  with 
the  liver  excluded  from  the  circulation,  bile  pigments  appear  in  the 
urine  and  icterus  is  manifested  in  the  fat  tissues,  from  which  observa- 
tions it  is  concluded  that  the  Hver  may  not  be  the  only  place  in  which 
bile  pigment  can  be  formed  from  hemoglobin.''"  Several  authors  have 
found  bilirubin  produced  in  hemorrhagic  effusions  located  where  the 
liver  could  have  had  no  influence. ^^  We  also  recognize  tj'pes  of  hemo- 
lytic icterus  in  which  the  liver  does  not  seem  to  be  concerned,  and 
with  bile  pigments  present  in  the  blood  and  urine  unaccompanied  by 
bile  salts  (dissociated  icterus),  so  that  the  old  dictum  of  the  essential 
implication  of  the  liver  in  icterus  seems  to  be  incorrect.-'-'  Joanno- 
vics^''  gives,  as  a  result  of  a  comparative  study  of  icterus  from  bile 
obstruction  and  icterus  from  hemolysis,   the  following  chief  differ- 

2«  See  Eppingcr,  Ziegler's  Beitr.,  1903  (33),  123;  Gerhardt,  Miiuch.  incd.  Woch., 
1905  (52),  889.  Lang  (Zeit.  exp.  Path.  ii.  Ther.,  July,  190G  (3),  473)  has  demon- 
strated the  presence  of  fibrinogen  in  the  bile  in  i)hosph()rus-poisoning,  which 
perhaps  accounts  for  the  "bile  thrombi"  ol)servod  l)y  Kppinger  in  toxic  icterus. 

"  See  Mendel  and  Underbill,  Amer.  Jour.  Phj^sioL,  1905  (14),  252;  Whipple 
and  King,  Jour.  Exp.  Med.,  1911  (13),  115. 

28  Sterling,  Arch.  exp.  Path.,  1911  ((U),  468;  Fiessinger,  Jour.  Physiol,  et 
Pathol.,  1910  (12),  958.  Oertel  (Arch.  Int.  Med.,  191S  (21),  73)  suggests  (hat 
intracellular  preciintation  of  bile  pigment  within  liver  cells  altered  by  poisons  may 
prevent  its  excretion  into  the  bile  canaliculi. 

-'■Mour.  Exi)er.  Med.,  1913  (17),  593  and  012. 

'"  Attempts  to  i)roduce  bile  pigments  from  hemoglobin  by  bacterial  action 
have  been  unsuccessful.      (()uadri,  Fol.  Clin.  Chim.,  1914,  No.  10.) 

="  Hooper  and  \Vhipi)le,  Jour.  Kx]).  Med.,  191()  (23),  137. 

'■>-  See  Leiu'liiic,  Ikntr.  i)atli.  Anat.,  1917  ((14),  55. 

'•■'  Zeit.  f.  llcilk..  Path.  Abt.,  1904  (25),  25. 


ICTERUS  491 

ences:  Icterus  due  to  hemolysis  appears  sooner  than  icterus  from 
bile-duct  occlusion,  and  reaches  a  much  higher  degree;  the  obstruction 
in  hemolytic  icterus,  when  present,  is  intra-acinous;  in  stasis  it  is 
chiefly  inter-acinous;  in  hemolytic  icterus  there  is  a  large  splenic 
tumor  due  to  accumulation  of  degenerated  red  cells  in  the  spleen, 
where  they  become  disintegrated  preliminary  to  the  formation  of  bile- 
pigment.  If  the  spleen  is  removed,  hemolytic  agents  may  not  cause 
icterus,  because  the  corpuscles  are  not  then  prepared  for  pigment 
formation. ^^  In  obstructive  icterus  from  gall  stones  there  is  a  choles- 
terolemia  in  proportion  to  the  amount  of  icterus,  which  is  not  usually 
true  of  icterus  from  other  causes." 

Toxicity  of  Bile. — In  any  event,  we  must  appreciate  that  in 
icterus  not  only  are  abnormally  large  quantities  of  bile-pigment  present 
in  the  blood,  but  also  usually  the  other  less  conspicuous  constituents 
of  the  bile.  Whole  bile  of  rabbits  is  fatal  to  rabbits  in  doses  of  0.2.5 
to  0.5  cc.  per  kilo,  by  intraperitoneal  injection,  and  about  half  as  much 
intravenously  (Bunting  and  Brown^").  Death  is  the  result  of  changes 
in  the  myocardium,  where  necrosis  is  produced;  and  severe  degenera- 
tive changes  are  also  found  in  the  kidneys  and  liver;  when  the  bile  is 
injected  into  the  peritoneum,  pancreatitis  and  fat  necrosis  result.  The 
relative  toxicity  of  the  bile-pigments  and  the  bile  salts  is  not  as  yet 
uniformly  agreed  upon. 

Bile-pigments. — Bouchard"  and  others  have  claimed  that  the  bile- 
pigments  are  far  more  toxic  than  the  bile  salts,  which  is  contradicted 
by  Rywosch  and  others.  Bihrubin  is  normally  present  in  the  blood, 
and  is  probably  responsible  for  the  yellow  color  of  the  plasma. ^^  It 
is  always  present  in  excess  in  icterus  of  whatever  degree. ^^  A  series 
of  analyses  by  Gilbert^"  and  others  gave  the  following  results:  Normal 
blood-serum  contains  0.027-0.08  gram  bilirubin  per  liter;  in  obstructive 
icterus  they  found  0.7  to  1.0  gram  of  bihrubin  per  hter,  in  biliary  cir- 
rhosis 0.33  gram  per  liter,  in  icterus  neonatorum  0.2  to  0.5  gram;  in 
pneumonia  0.068  gram  was  found.  These  figures,  however,  are  far 
in  excess  of  those  described  by  later  investigators.  Bauer  and  Spiegel"*^ 
give  figures  of  about  1  part  in  100,000  to  200,000,  or  0.01  to  0.005  gm. 
per  hter.  In  icterus  the  highest  figure  given  by  these  authors  was 
0.07  gm.  There  is  a  marked  variation  between  different  normal 
individuals,  but  for  the  same  person  the  figures  are  nearly  constant. 
The  threshhold  value  for  the  blood  seems  to  be  about  1  part  in  50,000; 

**  The  etiology  of  icterus  neonatorum  (when  not  obstructive)  has  not  been 
ascertained,  but  a  natural  tendency  towards  icterus  is  said  to  exist  in  the  new- 
born, their  blood  containing  much  more  bile  pigment  then  than  later.  (Hirsch, 
Zeit.  Kinderheilk.,  1913  (9),  196;  \lppa,  Miinch.  med.  Woch.,  1913  (39),  2161.) 

"  Rothschild  and  Felsen,  Arch.  Int.  Med.,  1919  (24),  520. 

3«  Jour.  Exper.  Med.,  1911  (1-4),  -145. 

"  Literature  and  discussion  by  Stadelmann,  Zeit.  f.  Biol.,  1896  (34),  57. 

3s  Blankenhorn,  Arch.  Int.  Med.,  1917  (19),  344;  1918  (21),  282. 

'9  Feigl  and  C^uerner,  Zeit.  exp.  Med.,  1919  (9),  153. 

"»  Compt.  Rend.  Soc.  Biol.,  1905  and  1906. 

^1  Deut.  Arch.  klin.  Med.,  1919  (129),  17. 


492  PATHOLOGICAL  PIGMENTATION 

when  this  proportion  is  exceeded  the  pigment  begins  to  be  deposited 
in  the  skin  and  excreted  by  the  kidneys  (v.  d.  Bergh),  However, 
Blankenhorn  believes  that  at  times  the  bihrubin  in  the  plasma  is  so 
bound  that  the  kidneys  do  not  excrete  it,  and  yet  it  may  be  able  to 
diffuse  into  the  skin.'*^  AVith  reduced  renal  function  the  amount  of 
pigment  in  the  blood  may  be  increased  without  hepatic  chsease. 

King  and  Stewart'*^  state  that  the  amount  of  pigment  in  a  lethal 
dose  of  whole  bile  will  cause  death,  but  the  bile  salts  present  in  the 
same  quantity  of  bile  will  not  cause  recognizable  effects;  uncombined 
pigment  is  more  toxic  than  its  calcium  or  magnesium  salts.  Bile  from 
which  the  pigment  is  removed  has  very  little  toxicity.  They  suggest 
that  calcium  is  increased  in  the  blood  in  icterus  as  a  protection  against 
the  toxic  effects  of  the  pigments.  The  combining  of  the  calcium  with 
bile  pigment,  however,  renders  it  unavailable  for  fibrin  formation,  and 
this  seems  to  be  an  important  factor  in  the  hemophilic  tendency  of 
icterus,'**  and  Pettibone  records  a  marked  decrease  in  blood  calcium 
in  protracted  jaundice.*^  The  decrease  in  available  calcium  may  also 
be  responsible  for  the  bradycardia  and  some  of  the  mental  and  nervous 
symptoms. 

Bile  salts  are  said  to  be  toxic,  generally  producing  depression 
of  the  central  nervous  system,  with  resulting  coma  and  paralysis; 
they  are  also  decidedly  toxic  to  cells  of  all  sorts,  causing  hemolysis  and 
marked  destruction  of  tissue-cells.  Small  quantities  of  bile  salts 
stimulate  the  central  end  of  the  vagus,  and  large  amounts  influence 
the  heart  itself;  hence  in  icterus  we  observe  a  slowing,  and  often  an 
irregularity,  of  the  pulse,  and  the  blood  pressure  is  lowered.  Al- 
though there  has  been  much  dispute  as  to  whether  the  chief  effects  of 
icterus  upon  the  heart  depend  upon  action  of  the  bile  salts  upon  the 
vagus,  or  upon  the  intracardiac  ganglia,  or  upon  the  muscle  itself,"'*' 
yet  Weintraud  demonstrated  that  in  some  cases  of  icterus  administra- 
tion of  atropin,  which  paralyzes  the  vagus,  stops  the  bradycardia, 
indicating  the  importance  of  the  effects  of  the  bile  salts  upon  the 
vagus  in  causing  this  feature  of  cholemia.  According  to  Meltzer  and 
Salant,*'''  bile  also  contains  a  tetanic  element  which  disajipears  from 
stagnating  bile;  the  bile  salts  contain  this  tetanizing  agent  in  less 
amount  than  does  the  whole  bile.  But  King'**  and  others  ascribe  most 
of  the  effects  of  bile  on  the  heart  to  the  bile  pigments,  perhaps  through 
abstraction  of  the  calcium.     Taurin  given  in  10  gm.  and  even  larger 

"  Corroborated  by  Meulengracht  (Ugesk.  f.  Laeger.,  1919  (SI),  1785)  who  states 
that  when  bilirubin  reaches  a  certain  concentration  it  passes  into  the  tissues,  but 
not  into  the  urine  until  a  higher  concentration  is  readied. 

"  Jour.  Exi)er.  Med.,  1909  (11),  07:i 

*"  See  Lee  and  Vincent,  Arch.  Int.  Med.,  1915  (16),  59. 

^i"  Jour.  Lab.  Clin.  Med.,  191S  (3),  275. 

"  See  Minkowski,  Ergel).  der  Pathol.,  1895  (2),  709. 

"  Jour.  Exp.  Med.,  1906  (8),  128;  review  and  literature  concerning  toxicity  of 
bile. 

"  Sec  King,  Bigelow  and  Pcarce,  Jour.  V]\\\n\  Med.,  1912  (14),  159. 


ICTERUS  493 

doses  by  mouth,  subcutaneously  and  intravenously  to  man  produced 
no  noticeable  effects  (Schmidt).'*^ 

Since  the  bile  salts  cause  hemolysis,  and  since  in  even  "hematogen- 
ous" jaundice  they  may  enter  the  blood,  it  can  readily  be  seen  that  in 
this  way  an  increased  formation  of  bile-pigment  may  be  incited  which 
leads  to  further  obstruction  to  the  outflow  of  bile  from  the  liver,  and 
a  "vicious  circle"  may  thus  be  established.  The  necroses  observed  in 
the  liver  in  icterus,  "icteric  necrosis,"  are  generally  ascribed  to  the 
cj'totoxic  effects  of  the  bile  salts,  although  it  is  difficult  always  to 
exclude  infection  extending  along  the  bile-ducts  to  the  liver  tissue. 
Possibly  the  power  of  bile  salts  to  dissolve  lipoids  may  be  responsible 
for  the  cytotoxic  effects^"  as  well  as  for  the  hemolysis.  The  itching 
and  irritation  of  the  skin  in  icterus  may  be  due  to  the  effect  of  the 
bile-salts  deposited  in  it,  for  pruritus  is  said  to  be  absent  in  the  pig- 
mentary jaundice  of  congenital  hemolytic  icterus.  There  is  also  an 
increase  in  the  cholesterol  in  the  blood,  which  may  be  related  to  the 
"xanthomas"  that  form  in  chronic  icterus. ^^  Unfortunately  we  have 
no  accurate  method  for  quantitative  determination  of  the  amount  of 
bile  salts  in  the  blood. 

A  remarkable  tendency  to  spontaneous  hemorrhages,  frequently  ob- 
served in  icterus,  probably  depends  upon  injury  to  the  capillary 
endothelium  by  the  bile  salts,  ^-  while  the  protracted,  often  uncontrol- 
lable, hemorrhage  that  may  occur  from  operation  wounds  in  icteric  pa- 
tients, is  related  to  the  slowed  coagulation  of  the  blood  observed  in 
icterus.  The  bile  salts  themselves  may  delay  coagulation  by  interfer- 
ing with  the  conversion  of  fibrinogen  into  fibrin. ^^  The  cytotoxic 
effect  of  the  bile  salts  is  also  shown  by  the  albuminuria  of  icteric  per- 
sons, which  frequently  results  from  the  renal  lesions  the  bile  produces. 
Although  bile  itself  is  toxic  to  many  bacteria,  especially  the  pneumo- 
coc.cus,'^'*  3^et  in  icterus  the  bactericidal  power  of  the  blood  is  lowered, 
and  infections  are  prone  to  develop  and  to  be  severe;  moreover,  the 
growth  of  several  species  of  bacteria  is  favored  by  bile.^" 

Croftan^''  summarizes  the  physiological  effects  of  bile  acids  as  fol- 
lows: (1)  A  powerful  cytolytic  action,  affecting  both  blood-cor- 
puscles and  tissue-cells.  (2)  A  distinct  cholagogue  action.  (3)  In 
small  doses  (1-500)  they  aid  coagulation.  (4)  In  large  doses  (1-250 
and  over)  they  retard  coagulation.  (5)  Slow  the  heart  action.  ^^ 
(6)  In  small  doses  they  act  as  vasodilators;  in  large  doses,  as  vaso  con- 

"  Schmidt  et  al,  Jour.  Biol.  Chem.,  1918  (33),  501. 

50  Neufeld  and  Handel,  Arb.  kaiserl.  Ges.-Amte,  1908  (28),  572. 

51  Chauffard,  Presse  Med.,  1913  (21),  81;  Chvostek,  Zeit.  klin.  Med.,  1911  (73), 
479;  Pinkus  and  Pick,  Deut.  med.  Woch.,  1908  (34),  1427. 

"  See  Morawitz.  Arch.  exp.  Path.,  1907  (56),  115. 
"  IIaes>ler  and  Stebbins,  Jour.  Exp.  Med.,  1919  (29),  445. 
*'*  See  Neufeld  and  Haendel,  loc.  cit. 
«  See  Meyerstein,  Cent.  f.  Bakt.,  1907  (44),  434. 

56  New  York  Med.  Jour.,  1906  (83),  810;  see  also  Faust,  "Die  tierische  Gifte," 
Braunschweig,  1906,  p.  29. 

"  See  Berti,  Gaz.  degli  Osped.,  1916  (37),  1233. 


494  PATHOLOGICAL  PIGMENTATION 

strictors.  (7)  Reduce  motor  and  sensory  irritability.  (8)  Act  on 
the  higher  cerebral  centers,  causing  coma,  stupor,  and  death.  Sel- 
lards^^  found  that  injection  of  bile  salts  into  guinea  pigs  causes  ulcer- 
ation and  hemorrhage  in  the  stomach. 

It  is  difficult  to  decide  how  much  of  the  profound  intoxication  that 
is  sometimes  present  in  icterus  ("cholemia"  and  "icterus  gravis") 
to  ascribe  to  the  reabsorbed  bile,  for  frequently  there  is  an  accompany- 
ing infection,  and  even  if  there  is  no  infection  the  impairment  of  liver 
function  by  the  obstruction  of  bile  outflow  must  also  be  reckoned 
with.  The  liver  is  not  only  the  great  destroyer  of  toxic  substances 
absorbed  from  the  alimentary  canal,  but  it  is  also  an  important  seat 
of  nitrogenous  metabolism,  interference  with  which  may  lead  to  ac- 
cumulation of  many  toxic  nitrogenous  substances  in  the  blood.  ^^  The 
long  duration  of  severe  icterus  in  some  cases  of  occlusion  of  the  bile- 
ducts,  with  relatively  shght  evidences  of  intoxication,  would  seem  to 
indicate,  however,  that  on  the  whole  the  bile  is  not  so  much  respon- 
sible for  the  intoxication  observed  in  icterus  as  are  the  associated 
conditions.  On  the  other  hand,  in  not  a  few  instances  it  has  been 
observed  that  escape  of  large  quantities  of  bile  into  the  peritoneal 
cavity  may  be  followed  by  symptoms  similar  to  those  of  icterus  gravis; 
in  these  cases  only  the  bile  can  be  held  responsible  for  the  intoxica- 
tion.«" 

Dissociated  Jaundice*^'  is  the  existence  of  cither  bile  salts  or  bile  pigment  sep- 
arately in  the  blood.  This  may  be  produced  either  by  the  bile  salts  being  ex- 
creted by  the  kidney,  leaving  only  the  less  diffusible  pigment  in  the  blood,  or  by 
separate  escape  of  bile  salts  from  the  liver  into  the  blood.  Also  in  true  hem- 
olytic icterus  we  may  have  bile  pigments  present  in  the  blood  without  bile  salts. 

Congenital  Hemolytic  Icterus.'^- — This  term  describes  a  condition  characterized 
by  a  chronic,  non-obstructive  jaundice,  without  evident  intoxication.  A  similar 
condition  is  also  observed  developing  in  adults,  without  familial  tendencies.  The 
congenital  form  usually  shows  familial  character,  but  isolated  congenital  cases  do 
occur.  It  is  the  result  of  active  hemolysis,  apparently  taking  place  chiefly  in  the 
spleen,  and  leading  to  an  icterus  without  evident  participation  of  the  liver.  The 
cause  of  the  hemolysis  is  entirely  unknown,  although  there  is  a  marked  fragility  of 
the  erythrocytes  evidenced  by  reduction  of  their  resistance  to  hypotonic  solutions, 
and  it  results  in  a  moderate  anemia,  with  excretion  of  much  uroliilin  in  both  stools 
and  urine;  the  blood  contains  biliru])in  which  is  not  excreted  in  the  urine.  The 
jaundice  is  usuall3^  unaccompanied  by  evidence  of  cholemia,  icteric  pruritus  or 
hemophilia.  The  spleen  is  greatly  enlarged  and  improvement  has  generally  fol- 
lowed splenectomy  but  the  exact  relation  of  the  spleen  to  the  disease  is  not  known." 
The  frequent  occurrence  of  gall  stones  in  this  condition  may  be  the  result  of  hyper- 
cholesterolemia from  hemolysis. 

The  metabolism  of  a  case*^'  showed  loss  of  nitrogen,  calcium,  magnesivnn  and 
iron,  and  a  much  increased  uric  acid  excretion.  These  conditions  may  improve 
after  operation.^^ 

"^  Arch.  Int.  Med.,  1909  (4),  502. 

'«  See  Bickel,  E.xper.  Untersuch.  iiber  der  Pathol,  der  Cholaemie,  Wiesbaden 
1900. 

«»See  Ehrhardt,  Arch.  klin.  Chir.,  1901  (64),  314. 

0' Hoover  and  Hlankenhorn,  Arch.  Int.  Med.,  1916  HS),  289. 

"  See  Richards  and  Johnson,  Jour.  Amer.  Med.  .Vssoc,  1913  (61),  1586. 

•3  See  series  of  articles  by  Pearce  el  al.,  in  Jour.  Exp.  Med.,  on  Relation  of 
Spleen  to  Blood  Destruction. 

6<  McKelvv  and  Rosenbloom,  Arch.  Int.  Med.,  1915  (15),  227. 

«  Goldschmidt,  Pepper  and  Pearce,  Arch.  Int.  Med.,  1915  (16),  437. 


UROBILIN  495 

The  Pigmentation  in  Icterus. — Living  tissues  have  but  a  slight 
tendency  to  take  up  bilc-pignicnts,  much  of  the  tissue-staining  ob- 
served at  autopsy  being  due  to  postmortem  imbibition  from  the  blood 
and  lymph.  Quincke*^®  found  that  after  subcutaneous  injection  of 
bihrubin  only  the  connective  tissue,  both  cells  and  intercellular  fibrils, 
becomes  diffusely  colored;  later,  it  fades  out  of  the  cells,  leaving  only 
the  fibrils  stained.  Muscle-cells,  fat-cells,  and  vessel-walls  take  up  the 
pigment  only  after  their  death.  If  the  jaundice  continues  for  a  long 
time,  the  subcutaneous  deposits  of  bilirubin  may  undergo  a  slow  oxida- 
tion, the  color  changing  to  an  olive  or  to  a  dirty  grayish  green.  The 
pigment  in  the  connective  tissues  is  at  first  in  solution,  but  may  be  de- 
posited in  a  granular  form  after  a  considerable  amount  has  accumu- 
lated. Bile  pigments  and  bile  salts  may  both  be  present  in  consider- 
able amounts  in  the  blood  and  not  pass  through  the  kidneys,  and  also 
they  may  fail  to  pass  into  the  tissues;  hence  we  may  have  cholemia 
without  icterus  or  choluria,  because  of  the  firmness  with  which  the  pig- 
ments are  bound  in  the  plasma  (Hoover^^). 

The  question  whether  in  icterus  the  skin  may  be  colored  b}'  other 
pigments  than  bilirubin,  especially  by  its  reduction  product,  urobilin, 
seems  to  have  been  decided  negatively.  Bile-pigment  is  probably  not 
absorbed  as  such  from  the  intestine  in  sufficient  quantity  to  cause 
icterus.  Such  bile-pigment  as  enters  the  blood  from  the  liver  is  ex- 
creted through  the  kidneys  chiefly,  but  also  in  the  sweat.  Ordinarily, 
other  secretions  (milk,  tears,  saliva,  sputum)  are  not  colored  in  jaun- 
dice, but  if  the  secretions  are  mixed  with  inflammatory  exudations, 
they  may  then  be  colored  (e.  g.,  pneumonic  sputum).  When  the  bile- 
pigment  is  resorbed  from  the  skin,  it  may  be  in  part  transformed  into 
urobilin,  which  appears  in  the  urine  in  increased  amounts  during  the 
period  of  recovery  from  jaundice.  Part  of  the  bile-pigment  is  prob- 
ably eliminated  by  the  liver  after  the  cause  of  obstruction  has  been 
removed  from  the  bile-passages. 

Urobilin" 

This  pigment  is  probably  formed  chiefly,  if  not  solely,  from  bile  pig- 
ments by  the  action  of  reducing  bacteria  in  the  intestine.  It  is  ex- 
creted in  the  urine  only  as  its  chromogen,  urobilinogen,  but  in  the 
feces  both  urobilin  and  urobihnogen  may  be  found;  when  exposed  to 
air  the  chromogen  oxidizes  quickly  to  urobilin.  Addis^*  states  that 
bilirubin  is  reduced  to  urobilinogen  in  the  bowel  and  is  then  largely 
absorbed,  to  be  at  once  oxidized  and  polymerized  into  urobilin,  two 
molecules  of  urobilinogen  uniting  under  the  influence  of  oxj^gen  to 
form  one  of  urobilin.     In  the  liver  the  urobilin  is  largely  worked  over 

««  Vichow's  Arch.,  1884  (95),  125. 

«^  Bibliography  and  review  by  Meyer-Betz,  Ergeb.  inn.  Med.,  1913  (12),  734; 
Wilbur  and  Addis,  Arch.  Int.  Med.,  1914  (13),  235. 
68  Arch.  Int.  Med.,  1915  (15),  412. 


496  PATHOLOGICAL  PIGMENTATION 

to  form  new  hemoglobin,  and  hence  the  functional  capacity  of  the 
liver  is  indicated  by  the  completeness  with  which  it  utilizes  the  uro- 
bilin, except  in  cases  of  excessive  formation  of  urobilinogen  as  a  re- 
sult of  hemolysis.  The  amount  of  urobilinogen  in  the  urine  will  be 
found  increased,  therefore,  in  hemolytic  icterus,  and  decreased  in  ob- 
structive icterus.  Exceptionally,  urobilinogen  may  be  formed  from 
blood  disintegrated  in  bloody  effusions  without  evident  participation 
of  the  liver,  e.  g.,  urobilinogenuria  with  hemorrhagic  ascites,  hemolytic 
poisons,  etc.  With  a  normal  liver  urobilinogenuria  is  found  only 
when  there  is  excessive  hemolysis,  otherwise  urobilinogenuria  occurs 
only  with  an  injury  to  the  liver  parenchyma  (Hildebrant).  In  general, 
the  amount  in  the  urine  is  an  index  of  the  amount  of  blood  destruction.^^ 
There  seems  to  be  little  if  any  retention  by  imperfectly  functioning 
kidneys  (Blankenhorn)  and  it  can  often  be  found  in  the  urine  when  not 
demonstrable  in  the  blood.  Occlusion  of  the  bile  ducts  stops  an  ex- 
isting urobilinogenuria  by  preventing  the  formation  of  urobilinogen 
in  the  intestine.  Normally  there  is  a  very  small  amount  of  urobilin- 
ogen and  related  substances  in  the  urine,  which  disappears  when 
there  is  no  bile  in  the  intestine.  Fromholdt^^  considers  that  increased 
■  bacterial  reduction  in  the  intestines  may  by  itself  account  for  uro- 
bilinogenuria. The  amount  of  urobilin  and  urobilinogen  excreted  in 
the  feces,  seems  to  vary  directly  with  the  amount  of  hemolysis, ^^  and 
the  same  is  true  for  the  duodenal  contents.'^'  The  evidence  of  abnor- 
mal hemolysis  is  said  to  occur  first  in  the  stools,  then  in  the  duo- 
denal contents,  and  lastly  in  the  urine;  the  presence  of  even  small 
amounts  of  urobilinogen  in  the  urine  being  evidence  of  a  probable  per- 
nicious anemia  in  the  absence  of  signs  of  biliary  and  hepatic  disease.^-" 

Digestive  Disturbances  in  Obstructive  Icterus." — In  case  the  icterus  depends 
upon  the  occlusion  of  the  main  bile-passages  by  stones,  tumors,  etc.,  the  situation 
is  complicated  by  the  effects  of  the  absence  of  this  natural  secretion  in  the  in- 
testinal canal.  Carbohydrate  and  protein  digestion  seem  to  be  but  little  affected, 
especially  the  former,  but  the  proportion  of  the  ingested  fat  that  appears  in  the 
feces  increases  from  the  normal  7-11  per  cent,  to  GO-<SO  per  cent.  The  products 
of  bacterial  decomposition  of  the  undigested  fat  may  lead  to  injury  of  the  in- 
testinal wall  and  disturbance  of  its  function.  Failure  of  absorption  of  fat  also 
favors  intestinal  putrefaction  by  enveloping  the  protein  substances  so  that  they 
are  not  readily  digested  and  absorbed.  The  relation  of  bile  to  intestinal  putre- 
faction is  still  not  exactly  determined.  Frequently,  but  by  no  means  always, 
there  is  an  increased  intestinal  putrefaction  which  may  result  in  diarrhea  and 
the  appearance  of  excessive  quantities  of  indican  and  phenol  in  the  urine.  The 
idea  once  held  that  the  bile  salts  acted  as  intestinal  antiseptics  has  not  been 
established  by  experimental  investigations;  however,  it  is  possible  that  through 
their  function  as  natural  cathartics,  by  stimulation  of  peristalsis,  they  prevent 
stagnation  and  putrefaction  of  proteins. 

»»  Dubin,  Jour.  Exp.  Med.,  1918  (28),  313. 
'0  Zeit.  exp.  Path.,  1911  (9),  268. 

71  Robertson,  Arch.  Int.  Med.,  1916  (15),  1072;  McCrudden,  Bost.  Med.  Surg. 
Jour.,  I!tl7  (177),  907. 

"  Gifhn,  Sanford  and  Szlapka,  Amer.  Jour.  Med.  Sci.,  1918  (155),  502. 
''''' Hausmann  and  Howard,  .lour.  Amer.  Med.  Assoc,  1919  (73),  1202 
"Concerning  metabolism  in  icterus  .:ee  Vannini,  Zeit.  klin.  Med.,  1912  (75),  136. 


CHAPTER  XIX 
THE   CHEMISTRY  OF  TUMORS^ 

Chemical  investigations  of  tumors  have  been  relatively  few  in  num- 
ber, but,  so  far  as  they  have  yet  been  made,  there  has  been  detected 
little  that  indicates  any  important  deviation  of  the  chemical  processes 
of  tumors  from  those  of  normal  cells  of  similar  origin.  Likewise,  the 
chemical  composition  of  tumor  tissue  resembles  closely,  on  the  whole, 
the  ('omposition  of  related  normal  tissues.  It  is  hardly  to  be  im- 
agined that  the  course  of  chemical  changes  is  greatly  different  in 
tumor  cells  from  that  in  normal  cells,  in  view  of  the  abundant  evi- 
dence that  the  metaboHc  products  of  tumor  cells  are  identical  with 
those  of  the  cells  from  which  they  arose.  Thus,  metastatic  growths 
of  thyroid  tissue  will  produce  thyroiodin  in  any  part  of  the  body,  liver 
carcinoma  metastases  produce  bile,  tumors  from  the  choroid  or  from 
pigmented  moles  produce  melanin,  etc.^  The  capacity  of  tumor  cells 
to  produce  complicated  products  of  metabolic  action  specific  for  the 
parent  cells  from  which  they  arose,  as  illustrated  above,  indicates 
beyond  question  that  the  course  of  their  chemical  activities  is  very 
much  like  that  of  normal  cells.  So,  too,  the  composition  of  the  cells 
is  found  to  be  similar  indeed  to  that  of  the  parent  cells,  both  in  re- 
gard to  primary  and  secondary  constituents.  Thus,  Bang  found  that 
sarcomas  derived  from  lymph-glands  contain  the  particular  nuclco- 
proteins  that  are  found  normally  only  in  Ijaiiph-glands,  hyperne- 
phromas contain  much  fat,  lecithin,  and  cholesterol;  squamous  cell 
carcinomas  develop  great  amounts  of  kerato-hyalin;  carcinomas  of 
mucous  membranes  may  contain  much  mucin,  etc. 

Many  have  sought  in  cancer  tissues  a  poison  that  might  account  for 
the  cachexia  characteristic  of  new-growths.  Extracts  have  been  ob- 
tained that  were  destructive  to  red  corpuscles  (hemolytic),  and  that 
were  sometimes  slightly  toxic  to  animals,  but  the  results  have  not 
seemed  sufficiently  striking  to  account  for  the  appearance  of  cachexia. 
Because  of  the  interference  with  circulation,  brought  about  in  tumors 
by  pressure  of  the  growing  tissues  upon  their  blood-vessels,  areas  of 
necrosis  frequently  develop,  and  these,  undergoing  autolysis,  yield  sub- 
stances that  are  hemolytic  and  toxic.  AVhether  these  are  the  cause  of 
cancer  cachexia,  however,  may  be  questioned;  but  they  are  sufficient 
to  account  for  most  of  the  experimental  results  as  yet  obtained.     No 

1  Earlier  literature  given  by  Neuberg,  Zeit.  Krebsforsch.,  1910  (10),  55;  and 
Blumenthal.  Ergebnisse  Physiol,  1910  (10),  363. 

2  See  Wells  and  Long,  Zeit.  Krebsforsch.,  1913  (12),  598. 

32  497 


498  THE  CHEMISTRY  OF  TUMORS 

substance  has  yet  been  isolated  from  or  detected  in  malignant  growths 
that  is  peculiar  to  them  and  not  found  in  normal  cells,  and  still  less 
has  any  substance  been  detected  that  accounts  in  an}^  way  either 
for  the  occurrence  of  tumors  or  for  the  effects  that  they  produce. 

Tumor  cells  seem  to  depend  upon  much  the  same  conditions  as  nor- 
mal body  cells  for  their  growth,  since  anything  that  leads  to  wasting, 
malnutrition,  or  atrophy  in  the  tissues  of  the  host  usually  tends  to 
impede  the  rate  of  growth  of  the  tumor  cells,  in  marked  contrast  to 
infectious  diseases.  Specific  attempts  to  modify  tumor  growths  by 
diets  (_Mendel-Osborne  diet)  which  stunt  the  animals  because  lack- 
ing certain  amino-acids  necessary  for  growth,  have  been  successful,^ 
but  it  is  cUfficult  to  be  sure  that  this  effect  depends  on  the  specific  ab- 
sence of  a  definite  substance  rather  than  on  general  malnutrition.'* 
Tumor  cells  made  incapable  of  utilizing  carbohydrate  through  com- 
plete phlorizin  diabetes^  may  be  unable  to  grow,  and  even  retro- 
gress completely.  Furthermore,  the  constituents  of  the  hypophysis 
that  stimulate  somatic  tissue  growth  are  also  said  to  stimulate  growth 
of  tumor  tissues.® 

The  discovery  by  B.  Fischer^  that  fat  stained  with  scarlet-R  and 
injected  beneath  the  skin  causes  epithelial  proliferation  resembling 
but  not  terminating  in  cancer,  has  led  to  much  speculation  as  to  the 
nature  of  substances  which  might  cause  cells  to  proliferate  lawlessly 
and  malignantly.^  The  great  frequency  of  cancer  in  workers  in  prod- 
ucts of  destructive  distillation  of  wood  (tar,  soot,  paraffin^)  has 
also  indicated  the  possibility  of  chemical  stimuli  causing  cancers.  A 
striking  instance  of  chemical  stimulation  causing  cancer  formation 
is  furnished  by  the  cases  of  carcinoma  of  the  urinary  bladder,  which 
is  a  common  cause  of  death  in  men  who  work  in  aniline  dyes,  both 
dyers  and  dye  makers  being  subject  to  this  condition.  The  dyes  that 
seem  to  be  responsible  belong  to  the  group  of  aromatic  amido-hy- 
droxyls,  including  safranin,  congo-red,  benzopurpurin,  fuchsin,  eosin 
and  others. ^°  Nassauer,^°"  however,  believes  that  the  aniline  itself 
is  the  active  agent.  H.  C.  Ross"  has  made  extensive  studies  of  the 
relation  to  cancer  of  substances  which  cause  leucocytes  to  multiply, 

=  See  Sweet,  etal,  Jour.  Biol.  Chem.,  1915  (21),  309. 

*  Rous,  Jour.  Exp.  Med.,  1914  (20),  433. 

5  Benedict  and  Lewis,  Proc.  Soc.  Exp.  Biol.,  1914  (11),  134. 

«  Robertson  and  Burnett,  Jour.  Exp.  Med.,  1916  (23),  631. 

'  Verb.  Deut.  Path.  Gcscll.,  1906  (10),  20;  sec  also  Haga,  Zeit.  Krebsforsch., 
1913  (12),  525;  Sachs,  Wicn.  klin.  Woch.,  1911  (24),  1551;  Stocber,  Munch,  med. 
Woch.,  1910  (57),  739  and  947. 

*  Stoeber  has  considered  substances  related  to  scarlet-R  that  might  have  this 
stimulating  effect,  and  found  naphthylaminol  most  active.  In  general,  fat-soluble 
organic  basic  substances  only  were  found  to  have  this  propertv,  anmng  them  being 
indole,  skatole  and  pyridine.  (Munch,  mod.  Woch.,  1909  (56),  129;  1910  (57), 
947). 

'■>  See  Bayon,  Lancet,  1912  (ii),  1579. 
'»  See  Leuenberger,  Beitr.  klin.  (^hir.,  1912  (SO),  20S. 
lo^Zeit.  angew.  Chem.,  1919  (32),  333. 
^'  "Researches  into  Induced  Cell  Reproduction  and  Cniioer,"  London.] 


PROTEINS  OF  TUMORS  499 

designating  them  as  "auxetics."  These  seem  to  be  present  in  the 
anthracene  fractions  of  tar,'-  wliich  may  explain  the  frequency  of  can- 
cer in  workers  in  tar,  soot  and  parafhn.  Japanese  investigators  report 
that  protracted  irritation  of  rabbits'  ears  with  tar  leads  to  strikingly 
infiltrative  proliferation  of  the  epithelium,  with  metastasis.^''  The 
influence  of  various  salts  on  cell  gi-owtli  has  also  been  applied  to  cancer 
pathology,  and  while  we  have  abundant  evidence  that  chemical  sub- 
stances may  either  stimulate  or  check  cell  growth,  as  well  as  regulate 
it,  our  biological  chemistry  has  not  yet  given  us  any  very  substantial 
facts  on  these  problems.^'* 

Nevertheless,  numerous  observations  have  been  made  concerning 
the  chemistry  of  tumors,  which,  although  they  do  not  as  yet  throw  any 
important  light  on  the  fundamental  problems  of  tumor  pathology, 
are  of  much  interest.     These  may  be  briefly  summarized  as  follows: 

A.  CHEMISTRY  OF  TUMORS  IN  GENERAL 

(1)  Proteins. — Earlier  studies  showed  that  tumor  growths  con- 
tain the  same  sorts  of  proteins  as  do  normal  tissues,  apparently  in 
about  the  same  proportions,  and  in  spite  of  certain  contradictor}-  re- 
ports this  statement  seems  to  be  correct. 

In  all  probability  the  nucleoproteins  of  tumors  share  the  specific 
characteristics  of  the  nucleoproteins  of  the  tissues  from  which  they 
arise — at  least  this  is  the  case  with  the  nucleoproteins  of  lymphosar- 
coma, according  to  Bang.'^  This  seems  to  have  been  confirmed  by 
Beebe,'^  who  found  nucleo-histon  only  in  lymph-gland  tissue,  but  the 
distinction  between  thymus  and  lymph-gland  nucleohiston  is  probably 
not  so  easily  made  as  Bang  intimates.  Because  of  their  richly  cellular 
structure  cancers  may  contain  more  nucleoprotein  than  the  tissues 
from  which  they  arise. ^^"  However,  Wells  and  Long'^  found  the 
proportion  of  purine  nitrogen  in  tumors  of  several  classes  to  be  much 
lower  than  might  be  expected  from  the  nuclear  content  as  shown  by  the 
microscope;  also,  Satta^^  found  unexpectedly  low  phosphorus  figures 
and  Yoshimoto^^  found  no  parallelism  between  the  number  of  nuclei 
and  the  nuclein  content.  The  purines  present  in  tumor  tissues  are 
quite  the  same  in  nature  and  proportion  as  in  normal  tissues  (Wells 
and  Long),  as  also  are  the  nucleoproteins. 

Bergell  and  D6rpinghaus-°  have  studied  the  nature  of  the  proteins 

'2  Norris,  Biochem.  Jour.,  1914  (8),  253. 
1'  Yamagiwa,  Mitt.  Med.  Gesellsch.,  Tokio,  1916  (30),  1. 

"  A  theory  of  cell  division  in  cancer  as  a  result  of  electric  forces  is  given  by 
Jessup  et  al,  Biochem.  Jour.,  1909  (4),  191. 
1*  Hofmeister's  Beitr..  1903  (4),  368. 
i^Amer.  Jour.  Physiol,  1905  (13),  341. 
i«»Petrey,  Zeit.  physiol.  Chem.,  1899  (27),  398. 
1"  Zeit.  f.  Krebsfrsch.,  1913  (12),  598. 
13  Arch.  Ital.  Biol.,  1908  (49),  380. 
'5  Biochem.  Zeit.,  1909  (22),  299. 
"  Deut.  med.  Woch.,  1905  (31),  1426. 


500  THE  CHEMISTRY  OF  TUMORS 

in  tumors  by  determining  the  proportion  of  the  various  amino-acids 
that  compose  them.  Because  of  the  amount  of  material  necessary 
for  the  ester  method,  they  were  obhged  to  use  a  mixture  of  various 
primary  and  secondary  cancers  and  one  sarcoma.  The  protein  of 
this  tumor-mixture  was  characterized  by  the  very  high  proportion  of 
alanine,  glutaminic  acid,  phenylalanine  and  aspartic  acid,  there  being 
from  5  to  10  per  cent,  of  each.  Leucine  was  very  low,  5-10  per  cent., 
as  against  20  per  cent.,  or  higher,  found  in  most  normal  tissues.  Gly- 
cine and  tyrosine  were  present  in  small  quantities,  and  serine  was  prob- 
ably also  present.  Neuberg^^  found  in  cancer  protein  1.3  per  cent, 
of  tyrosine,  17  per  cent,  of  leucine,  scarcely  1  per  cent,  of  glutaminic 
acid,  and  4.92  per  cent,  of  glja^inc.  In  five  human  tumors  of  different 
sorts,  Kocher--  found  high  figures  for  diamino-nitrogen;  his  averages 
were:  arginine,  12.42;  histidine,  4,86;  lysine,  11.23;  total,  28.47 
per  cent,  of  the  protein  nitrogen.  Drummond's'^  careful  studies  in 
this  field  have  shown  that  the  diamino-acid  content  of  tumors  is 
generally  slightly  higher  than  in  corresponding  normal  tissues,  prob- 
ably varying  directly  with  the  amount  of  nuclear  material,  there  being 
nothing  found  to  indicate  that  the  hexone  bases  are  in  any  way  respon- 
sible for  increased  growth.  Strange,  and  as  yet  unexplained,  varia- 
tions in  tryptophane  content  in  various  tumors  were  found  by  Fasal,-"* 
some  having  a  very  high  try{)tophane  figure,  while  in  others  none  could 
be  found.  Centanni"  found  that  trjqitophanc  and  tyrosine  inhibit, 
while  skatolo  and  indole  stimulate  carcinoma  growth. 

Certain  authors  have  believed  that  the  cancer  cell  has  a  specific 
chemistry,-*^  but  most  of  these  analyses,  including  that  of  Abderhalden 
and  Mcdigreceanu,-^  seem  to  indicate  that  cancer  proteins  have  much 
the  same  composition  as  normal  proteins.  Cramer  and  Pringle-^ 
find  that  there  is  less  nitrogen  in  mouse  cancers  than  in  equal  amounts  of 
other  mouse  tissue,  the  decrease  being  in  the  coagulable  nitrogen, 
incoagulable  nitrogen  being  relatively  increased;  a  given  amount  of 
nitrogen  produces  more  cancerous  than  normal  tissue.  The  water 
content  of  rapidly  growing  tissues,  whether  normal  or  cancerous,  was 
found  to  be  high.  This  corresponds  with  the  analysis  of  Robin,-' 
who  found  the  water  content  high  and  nitrogen  low  in  carcinomas  of 
the  liver,  suli)hur  being  especially  low,  and  C^hisholnr*"  has  found  the 
proportion  of  nitrogen  in  several  human  tumors  lower  than  in  the  soma- 

"^^  .\rb.  a.  d.  Path.  Inst,  zu  Berlin,  1906,  p.  593. 
"  Jour.  Biol.  Cheni.,  1915  (22).  295. 
"Biochem.  Jour.,  1910  (10),  473. 
^^  Biocheni,  Zeit.,  1913  (55),  88. 
"  Tumori,  1913  (2),  406. 

-»  Blumenthal,  Zeit.  Krebsforsch.,  1907  (5),  183. 
-'  Zeit.  phvsiol.  Cliem.,  1910  (09),  00. 

=«  I'roc.  Koval  Soc,  B.,  1910  (S2),  315;  Jour.  Phvsiol.,  1910  (50),  322. 
■'"Cent.  Phvs.  Path.  StotYwechs.,  1911  (0).  577.     Bull.  .Vcad.  M6J.,  1919  (81), 
799;  Coinpt.  Rend.  Acad.  Sci.,  1919  (108),  1071. 
"«  Jour.  Pathol,  and  Bact.,  1913  (17),  000. 


CARBOHYDRATES  OF  TUMORS  501 

tic  tissue.  However,  the  lack  of  any  marked  specific  individuality 
of  cancer  proteins  when  tested  by  immunological  reactions,  indicates 
a  very  close  chemical  agreement  with  normal  tissue  proteins. ' 

On  account  of  the  amount  of  autolysis  going  on  in  tumors  the 
products  of  protein  splitting  are  usually  present.  Beebe'''  found  in 
a  number  of  tumors  leucine,  tyrosine,  tryptophane,  proteoses  (biuret 
reaction),  and  in  one  glycine.  Drummond"  has  found  leucine, 
tyrosine  and  creatinine  commonly  present  in  water  extracts  of  malig- 
nant tumors.  Because  of  the  deficient  circulation  in  the  tumors,  the 
amino-acids  accumulate  in  the  cancer  tissues  in  sufficient  amounts 
to  be  detected,  and  may  be  found  even  when  no  macroscopic  evidences 
of  degeneration  are  present.  Possibly  on  account  of  this  poor  absorp- 
tion no  proteoses,  peptones,  or  amino-acids  could  be  found  in  the  urine 
of  cancer  patients  by  Wolff  ;''^  but  Ury  and  LilienthaP-*  found  a  posi- 
tive reaction  for  albumose  in  the  urine  in  about  two-thirds  of  all  car- 
cinoma cases  examined  by  them;  however,  it  may  be  absent  even  in 
advanced  stages.  Lactic  acid  is  also  present  in  tumors,  according  to 
Fulci^^  and  Saiki,^^  the  latter  finding  0.48  gm.  of  lactic  acid  per  100 
gms.  cancer  of  the  stomach.  According  to  Clowes"  cancer  tissues 
are  much  more  permeable  to  ions  than  are  normal  tissues. 

(2)  Other  Organic  Constituents. — These,  in  general,  resemble 
the  organic  constituents  of  the  tissue  from  which  the  tumor  arises,  for 
a  structural  resemblance  to  the  parent  tissue  always  exists,  and  as 
structural  features  depend  largely  on  the  proportion  of  the  chemical 
components,  a  structural  similarity fairh' implies  a  chemical  similarity. 
For  example,  adrenal  and  renal  tissue  contain  much  lecithin  and  choles- 
terol, and  hypernephromas  show  a  similar  composition;  the  fat  of  a 
lipoma  is,  in  its  qualitative  features,  almost  identical  with  the  normal 
fat  of  the  same  individual;  tumor  melanin  shows  no  characteristic 
chemical  distinction  from  normal  melanin,  etc. 

Glycogen  has  been  particularly  studied  in  tumors,  especially  be- 
cause of  the  erroneous  idea  advanced  by  Brault  that  the  quantity  of 
glycogen  is  in  direct  proportion  to  the  mahgnancy.  From  a  sum- 
mary of  all  the  evidence,  it  seems  that  two  chief  factors  determine  the 
presence  and  amount  of  glycogen  in  tumors.  One  is  the  embryonic 
origin  of  the  tumors;  thus  tumors  of  cartilage,  striated  muscle,  or  of 
squamous  epithelium,  which  tissues  normally  contain  much  glycogen, 
are  hkewise  provided  with  an  abundance  of  this  material.  Second, 
the  occurrence  of  areas  of  impaired  cell-nutrition  favors  the  accumu- 
lation of  glycogen  in  the  degenerating  tumor-cells  just  as  it  leads  to 

31  Amer.  Jour.  Physiol,  1904  (11),  139. 
«  Biochem.  Jour.,  1917  (11),  246. 

35  Zeit.  f.  Krebsforschung,  1905  (3),  95. 
3*  Arch.  f.  Yerdauungskr.,  1905  (11),  72. 
"  Gaz.  internaz.  dimed.,  1910,  No.  24. 

36  Arch.  m^d.  exp^r.,  1911  (23),  376. 

37  Proc.  Soc.  Exp.  Biol.  Med.,  1918  (15),  107. 


502  THE  CHEMISTRY  OF  TUMORS 

a  similar  accumulation  in  all  other  tissues  (Gierke). ^^"  The  most  ex- 
tensive consideration  of  this  topic  is  reported  by  Lubarsch,^^  who 
found  glycogen  microscopically  in  447  (or  29  per  cent.)  of  1544  tumors 
examined.  It  was  present  in  but  3  out  of  184  fibromas,  osteomas, 
gliomas,  hemangiomas,  lipomas,  and  lymphangiomas,  and  in  but  2 
out  of  260  adenomas  from  various  parts  of  the  body.  It  occurred  in 
all  teratomas,  rhabdomyomas,  hypernephromas,  and  syncytiomas. 
In  138  sarcomas  glycogen  was  present  in  70  (50.7  per  cent.);  of  415 
carcinomas  it  was  found  in  181  (43.6  per  cent.).  In  the  squamous 
epithelial  cancers  70  per  cent,  contained  glycogen,  while  the  mucoid 
or  colloid  cancers  were  always  free  from  glycogen.  The  glycogen 
undoubtedly  enters  the  cells  from  without,  probably  entering  as  sugar, 
and  being  converted  into  glycogen  by  intracellular  enzymes.  We  have 
no  rehable  studies  of  the  actual  quantity  of  glycogen  in  various  tumors, 
although  Meillere^^  states  that  the  microscopic  and  chemical  examina- 
tion of  tumors  give  corresponding  comparative  results,  which  Gierke 
states  is  generally  true  with  glycogen  estimations. 

Pentoses. — Neuberg'*'^  reports  finding,  as  a  product  of  autolysis  of  a 
carcinoma  of  the  liver,  a  pentose  which  was  not  produced  by  autolysis 
of  either  normal  liver  tissue  or  the  primary  growth  in  the  stomach. 
Beebe*^  found  that  in  carcinoma  of  the  mammary  gland  the  percentage 
of  pentose  (xylose)  is  somewhat  higher  than  the  amount  in  normal 
mammary  glands  f about  0.23  per  cent.).  Carcinoma  in  the  hver  did 
not  show  any  constant  excess  of  pentose  above  that  of  normal  liver 
tissue  (about  0.38  per  cent.).  A  primary  carcinoma  of  the  liver 
showed  quite  the  same  pentose  and  phosphorus  content  as  normal 
liver  tissue.  In  general,  no  constant  relation  of  pentose  to  origin, 
mahgnancy,  or  degeneration  of  tumors  was  observed. 

Purines  and  Purine  Enzymes. — ^The  purines  of  both  benign  and 
malignant  tumors  have  been  studied  by  Wells  and  Long, ''-  who  found 
them  the  same  as  those  in  normal  tissues,  and  in  much  the  same  rela- 
tive proportions.  The  proportion  of  the  total  nitrogen  of  tumors 
which  is  constituted  by  the  purine  nitrogen  is  less  than  would  be  ex- 
pected from  the  histological  evidence  of  the  amount  of  nuclear  material 
contained  in  the  tumors.  Tumors  also  seem  to  contain  much  the 
same  purine  enzymes  as  the  normal  tissues.  Thus,  guanase  seems  uni- 
versally present  in  tumors  derived  from  human  tissues,  and  adenase 
is  missing,  although  autolyzing  tumors  can  disintegrate  their  nucleic 
acid  (nuclease)  and  change  the  adenine  radicals  of  the  nucleic  acid 
into  hypoxanthine,  presumably  by  way  of  adenosine  and  inosine  ( Am- 
berg  and  Jones).     Secondary  tumors  growing  in  the  human  liver  do 

"«  Ziegler's  Beitr.,  1905  (.37),  502. 

'8  Virchow's  Arch.,  190G  (183),  188. 

3»  Coinpt.  Rend.  Hoc.  Biol.,  1900  (52),  324. 

"  Berl.  klin.  Woch.,  1904  (41),  1081;  1905  (42),  118. 

"'  Arncr.  Jour.  Physiol,  1905  (14),  231. 

*■'  Zeit.  f.  Krebsforsch.,  1913  (12),  598. 


LIPINS  OF  TUMORS  503 

not  acquire  the  enzyme,  xanthine-oxidase,  which  is  a  characteristic 
enzyme  of  this  organ.  Tlie  hver  tissue  between  the  cancer  nodules 
seems  to  oxidize  purines  less  actively  then  normal  liver  tissue.  Long-*' 
has  also  found  similar  conditions  in  tumors  from  sheep,  pigs  and  cattle, 
observing  that  primary  carcinoma  of  the  liver  does  not  contain  xan- 
thine oxidase,  a  point  of  interest  in  view  of  the  fact  that  in  the  develop- 
ment of  mammals  the  xanthine  oxidase  does  not  appear  until  late. 
Water  extracts  from  various  tumors  have  been  found  to  contain  small 
amounts  of  free  purines,  chiefly  adenine,  guanine  and  hypoxanthine 
(Drummond).''- 

Lipins. — Tumor  cells  seem  to  contain  much  the  same  fats  and  lip- 
oids as  normal  cells,  and,  as  far  as  known,  in  much  the  same  proportions 
as  characterize  the  cells  from  which  the  tumors  arose.  Thus  Wells^* 
found  that  hypernephromas  show  the  same  high  proportions  of  lecithin 
and  cholesterol  as  he  found  in  normal  adrenal,  and  as  are  found  in 
the  renal  cortex.  Other  malignant  tumors  have  much  less  lipoids  and 
fats(  see  Hypernephromas).  A  secondary  carcinoma  of  liver  cells, 
metastatic  in  the  skull,  was  found  by  Prym^"  to  show  the  same  sort 
of  fatty  infiltration  that  is  characteristic  of  fatty  liver  cells.  On  ac- 
count of  the  poor  blood  supply  of  many  tumors,  fatty  changes  are 
usual,  occurring  under  the  same  conditions  and  showing  the  same 
microscopic  features  as  fatty  degeneration  in  other  tissues,''*'  being 
more  common  in  malignant  than  in  benign  tumors;  especially  abund- 
ant in  squamous  cell  carcinomas,  and  scanty  in  sarcomas.  Crystals 
of  cholesterol  or  cholesterol  compounds  are  described  in  tumors  by 
White. '*^  Dewey**  found  the  chief  lipoid  in  jaw  tumors  to  be  choles- 
terol, with  more  or  less  free  fatty  acids  and  soaps,  according  to  mi- 
crochemical  determinations.  Even  lipoma  fat  shows  no  difference 
from  normal  fat,"*^  and  the  depot  fat  of  tumor  patients  is  quite  the  same 
as  in  patients  with  other  diseases  associated  with  equal  wasting,  *° 
in  whom  some  increase  in  unsaponifiable  material  (cholesterol)  is  usual. 
Murray^^  says  that  the  lipoids  of  degenerating  uterine  fibroids  are 
strongly  hemolytic,  which  may  account  for  the  so-called  ''red  degen- 
eration" of  these  tumors.  Freund  and  Kaminer^-  suggest  that  the 
fatty  acids  of  tissues  are  of  importance  in  determining  whether  a  tissue 
is  a  suitable  soil  for  secondary  growth,  these  substances  being  deficient 
in  tissues  where  growths  develop.  Mitochondria,  which  seem  to  be 
closely  related  to  the  intracellular  lipins,  show  no  constant  differences 

"  Jour.  Exper.  Med.,  1913  (18),  512. 
*"■  Jour.  Med.  Res.,  1908  (17),  461. 
«  Frankf.  Zeit.  Path.,  1912  (10),  170. 

«  See  Haga,  Berl.  klin.  Woch.,  1912  (49),  342;  Joannovics,  Wien.  klin.  Woch. 
1912  (25),  37. 

*"  Jour.  Path,  and  Bact.,  1908  (13),  3. 

*8  Jour.  Cancer  Res.,  1919  (4),  263. 

"  See  Wells,  Arch.  Int.  Med.,  1912  (10),  297. 

*»  Wacker,  Zeit.  physiol.  Chem.,  1912  (78),  349;  1912  (80),  383. 

°i  Jour.  Obst.  Gyn.  Brit.  Emp.,  1910  (17),  534. 

"  Wien.  klin.  Woch.,  1912  (25),  1698. 


504  THE  CHEMISTRY  OF  TUMORS 

in  cancer,  benign  tumors  and  normal  cells,  except  that  sometimes  in 
cancer  they  fix  stains  less  firmly  (^Goodpasture). ^^ 

There  has  been  some  effort  to  correlate  the  cholesterol  and  lecithin 
contents  of  blood  and  tissues  with  the  rate  of  cancer  growth;  apparent- 
ly lecithin  inhibits  growth  and  cholesterol  stimulates.^''  However, 
Bullock  and  Cramer"  found  much  more  cholesterol  in  a  slowly  growing 
mouse  carcinoma  than  in  a  rapidly  growing  one,  somewhat  more  phos- 
phatid  in  the  latter,  much  more  phosphatid  in  a  sarcoma  than 
in  the  carcinoma,  and  cerebrosides  only  in  the  latter;  in  necrotic 
portions  of  tumors  they  found  an  increase  in  simple  fats.  These 
figures  are  based  on  too  few  observations  to  be  interpreted  as  yet. 

(3)  Inorganic  Constituents. — These  have  been  studied  by  Clowes 
and  Frisbie^*^  under  exceptionably  favorable  conditions,  in  that  the  age 
of  the  tumor  could  be  accurately  estimated,  in  the  inoculable  carcinoma 
of  mice.  They  found  that  rapidly  growing  tumors  contain  a  high 
percentage  of  potassium  and  little  or  no  calcium,  whereas  in  old, 
slowly  growing,  relatively  necrobiotic  tumors  the  relation  is  reversed, 
the  potassium  decreasing  greatly  while  the  calcium  increases.  Mag- 
nesium is  present  only  in  traces,  while  the  proportion  of  sodium  fluc- 
tuates much  less,  but  is  usually  greater  than  either  the  potassium  or 
calcium,  although  in  very  old  tumors  the  latter  may  become  excessive. 
The  most  rapid  growth,  however,  seems  to  occur  in  tumors  in  which 
both  calcium  and  potassium  are  present  in  the  ratio  of 

K       2      3 
Ca  =  l°^'2 

Beebe^^  analyzed  a  number  of  human  tumors  with  the  following 
results:  Phosphorus  was  found  in  proportion  to  the  amount  of  nu- 
clear material,  varying  from  0.139  per  cent,  (uterine  fibroid)  to  1.06 
per  cent,  (sarcoma).  Iron  varied  from  0.013  per  cent,  to  0.064  per 
cent.,  probably  depending  on  the  amount  of  blood  and  nucleoproteins. 
Calcium  is  most  abundant  in  old  degenerated  tumors,  and  potassium 
in  rapidly  growing  tumors.  These  results,  supported  by  Clowes  and 
Frisbie's  findings,  indicate  the  importance  of  potassium  for  cell  growth. 
Injection  of  potassium  salts  into  mice  increases  their  susceptibility 
to  inoculation  (Clowes),^^  while  calcium  decreases  cancer  growth 
(^Goldzieher).^^  Exposure  of  isolated  cancer  cells  to  calcium  salts 
reduces  their  growth  capacity  when  inoculated,  apparently  through 
reducing  their  water  content;  both  effects  arc  counteracted  by  sodium 

"  Jour.  Med.  Res.,  1918  (38),  213. 

"  See  Robertson  and  Burnett,  Jour.  Exp.  Med.,  1913  (17),  3-14;  1016  (23), 
631;  Sweet  et  al,  Jour.  Biol.  Chem.,  1915  (21,,  309;  Luden,  Jour.  Lab.  Clin.  Med., 
vols.  3  and  4. 

^f-  Proc.  Royal  Soc.  London  (B),  1914  (87),  230. 

•■sAmer.  Jour.  Phvsiol.,  190.5  (14),  173. 

"  Amor.  Jour.  Pliysiol.,  1904  (12),  167. 

"  British  Med.  Jour.,  Doc.  1,  1906. 

6"  Vorhandl.  Dout.  Path.  Gesellsch.,  1912  (15),  283. 


I 


ENZYMES  OF  TUMORS  505 

(Cramer).*"  A  greater  proportion  of  potassium  was  found  in  primary 
than  in  secondary  growths  by  Mottram;'''  sodium  was  the  same  in 
each;  there  is  more  potassium  in  squamous  cell  carcinoma  than  in 
round  cell  sarcoma.  Robin*^'-  states  that  in  cancerous  livers  the  cancer 
tissues  contains  more  inorganic  matter  than  the  normal  liver  tissue 
about  it.  Cattley*'  found  the  microchemic  distribution  of  potassium 
the  same  in  cancer  as  in  normal  cells,  and  the  same  seems  to  be  true  of 
manganese.*^ 

Schwalbe''^  found  that  cancer-cells  contain  iron  in  a  condition  de- 
monstrable by  the  Berlin-blue  reaction,  and  occurring  independent 
of  hemorrhages.  Tracy^®  found  that  tumors  reacted  microscopically 
for  iron,  either  free  or  in  the  form  of  an  albimiinate,  only  in  areas  where 
hemorrhages  had  occurred.  Nuclear  or  organic  iron  could  be  detected 
in  the  nuclei,  occurring  in  a  network  arrangement.  In  other  words, 
iron  occurs  in  tumors,  both  quantitatively  and  qualitatively,  exactly 
as  in  normal  cells  of  the  same  type.  The  same  writer"  found  in  tumors 
by  microchemical  reactions,  that  phosphorus  in  the  form  of  nucleo- 
proteins  likewise  shows  no  essential  differences  from  its  distribution 
in  normal  tissues. 

In  this  connection  may  be  mentioned  the  observations  of  Hem- 
meter,***  who  found  that  the  cells  of  carcinoma  of  the  mammary  gland 
will  shrink  when  placed  in  physiological  salt  solution  or  in  the  serum 
of  the  patient,  whereas  normal  cells  swell  when  placed  in  cancer-juice. 
This  suggests  that  the  osmotic  pressure,  and,  by  inference,  the  amount 
of  inorganic  constituents,  is  lower  than  in  normal  tissues.  Crystal- 
loids, such  as  KI,  diffuse  readily  into  cancer  tissue.*^ 

(4)  Enzymes. — The  rapid  and  extensive  autolysis  that  occurs  in 
tumors,  as  shown  both  morphologically  and  by  the  presence  of  the 
products  of  protein  cleavage  in  them,  indicates  that  tumor  cells 
resemble  all  other  cells  in  possessing  intracellular  proteolytic  enzj^mes. 
Because  of  autolysis,  puncture  fluids  in  cancer  of  serous  surfaces  show 
an  increased  amount  of  incoagulable  nitrogen  (J\Iorris),^°  and  they 
may  show  free  amino-acids  ( Wiener), ^^  while  there  is  a  slight  increase 
in  the  incoagulable  nitrogen  of  the  blood  (Takemura).^- 

There  is  considerable  but  not  undisputed  evidence  that  cancer  tis- 

s"  Biochem.  Jour.  1918  (12).  210. 

"  Arch.  Middlesex  Hospital,  1910  (19),  40. 

«2  Compt.  Rend.  Acad.  Sci.,  1913  (156),  334. 

^^  Lancet   1907  (172)    13. 

«^  Medigreceanu,  Pro'c.  Royal  Soc,  B,  1912  (86),  174. 

"  Cent.  f.  Path.,  1901  (12),  874. 

66  Jour.  Med.  Research,  1905  (14),  1. 

"  Martha  Tracy,  Jour.  Med.  Research,  1906  (14),  447. 

68  Amer.  Jour.  Med.  Sci.,  1903  (125),  680. 

69  Van  den  Velden,  Biochem.  Zeit.,  1908  (9),  54;  see  also  Wells  and  Heden- 
burg.  Jour.  Infect.  Dis.,  1912  (11),  349. 

'"Arch.  Int.  Med.,  1911  (8),  457. 
'1  Biochem.  Zeit.,  1912  (41),  149. 
"  Ibid.,  1910  (25),  505. 


506  THE  CHEMISTRY  OF  TUMORS 

sue  autolyzes  somewhat  more  rapidly  than  corresponding  normal  tis- 
sues," and,  according  to  Neuberg,  Blumenthal  and  others,^'*  that  cancer 
extracts  digest  other  tissues  than  themselves  (heterolysis),  a  property 
not  exhibited  by  extracts  of  normal  tissues.  Miiller  and  others  would 
ascribe  this  heterolysis  to  the  leucocytes  present  in  the  tumors.  Nu- 
cleases have  been  found  in  tumors  as  in  other  tissues,"  and  in  general 
the  enzymes  which  deamidize  adenine  and  guanine  (adenase  and  gua- 
nase)  are  usually  present  if  the  original  tissue  possessed  these  enzymes 
but  no  instance  of  the  presence  of  xanthine  oxidase  or  uricolytic  en- 
zyme has  been  obtained  (Wells  and  Long,  loc  cit^'^). 

Hamburger  finds  that  the  enzymes  of  cancer  tissue  upon  which  the 
glycyl-tryptophane  and  other  enzyme  tests  for  cancer  are  based,  are 
ereptases,  resembling  in  all  their  properties  the  ereptases  of  normal 
tissues,  and  not  present  in  particularly  large  amount.  However,  Ab- 
derhalden^^  has  found  evidence  that  certain  peptids  may  be  split  in  a 
different  way  by  cancer  than  by  normal  tissues,  supporting  those  who 
hold  that  cancer  enzymes  are  different  from  normal  tissue  enzymes. 
Autolysis  of  tumors  is  said  to  be  augmented  by  x-ray,  and  especially 
by  radium  (Neuberg),  and  tumor  tissue  is  readily  digested  by  trypsin. 

The  presence  of  ereptases  in  carcinomatous  gastric  juice  has  been 
especially  studied  because  of  its  diagnostic  possibilities,  and  the  care- 
ful investigation  of  Jacques  and  Woodyatt''^  seems  to  show  conclu- 
sively that  such  an  enzyme  is  rarely  present  in  gastric  juice  except 
when  derived  from  a  cancer  present  in  the  wall  of  the  stomach,  pro- 
vided peptolytic  bacteria  are  excluded  by  filtration.  Deaminizing 
enzymes  may  also  be  found  in  gastric  cancer  secretions.''^  In  the  blood 
of  cancer  patients  there  is  usually  an  increased  antitryptic  activity, 
ascribable  to  the  reaction  against  enzymes  absorbed  from  the  cancer; 
it  is  less  pronounced  with  sarcoma.''^  The  body  tissues  of  patients 
dying  with  cancer  show  a  low  ereptic  activity,  but  the  same  occurs  in 
persons  dying  from  other  wasting  diseases  (Col well). ^^  This  is  also 
true  of  other  tissue  enzymes; — at  least  purine  oxidizing  enzymes  are 
deficient  in  the  liver  tissue  between  secondary  cancers  (Wells  and 
Long''-)  and  the  catalase  is  also  reduced  in  liver  tumors  (Blumenthal 
and  Brahn)^^  and  in  the  blood  of  tumor  mice  (Rosenthal);'^-  in  human 
blood  the  catalase  may  vary  either  side  of  normal.*^*     Brahn'^'*  reports 

'« See  Yoshimoto,  Biochem.  Zeit.,  1909  (22),  299;  Daels  and  Delenz6,  Bull. 
Acad.  Med.  Belg.,  1913  (26),  833. 

'^  Bibliography  by  Hamburger,  Jour.  Amer.  Med.  Assoc,  1912  (59),  847. 

'5  Goodman,  Jour.  Exp.  Med.,  1912  (15),  477. 

'«  Zeit.  Krebsforsch.,  1910  (9),  266. 

"  Arch.  Int.  Med.,  1912  (10),  560. 

'»  Halpern,  Mitt.  Grenz.  Med.  Chir.,  1915  (28),  709. 

'»  Citronblatt,  Med.  Klin.,  1912  (8),  138. 

80  Arch.  Middlesex  Hosp.,  1909  (15),  96. 

*'  Zeit.  f.  Krebsforsch.,  1910  (8),  436.  See  also  Weidenfeld,  Wien.  klin.  Woc-h., 
1918  (31),  324. 

82  Deut.  med.  Woch.,  1912  (38),  2270. 

8'  Rohdenburg,  N.  Y.  Med.  Jour.,  1913  (97),  824. 

»*Zeit.  Krebsforsch.,  1917  (16),  112. 


INTERNAL  SECRETION  OF  TUMORS  507 

that  the  hvcr  tissue  between  sccoiuhiry  cancer  nodules,  and  also  the 
liver  in  cases  of  cancer  in  the  portal  area,  shows  diminished  catalase, 
lipase  and  lecithinase  function,  with  increased  autolysis,  but  these 
changes  are  not  observed  in  the  livers  when  the  cancer  is  in  other  parts 
of  the  body.  However,  chol'ne  has  been  found  in  necrotic  sarcomas 
of  rats,^^  whicli  would  seem  to  indicate  the  presence  of  enzj'ines  dis- 
integrating lecithin.  As  mentioned  elsewhere  (See  Melanin),  me- 
lanotic tumors  may  contain  enzymes  oxidizing  tyrosine,  epinephrine, 
pyrocatechin,  or  other  related  aromatic  substances,  with  the  forma- 
tion of  pigmentary  substances.  (See  also,  Autolysis  in  Tumors, 
chap,  iii.) 

Other  enzymes  are  also  present  in  tumor  cells.  Buxton^*^  exam- 
ined a  large  number  of  tumors  for  their  enzymes  by  the  plate  {auxan- 
ographic)  method,  and  found  considerable  variations  in  different 
growths.  All  contained  amylase  (splitting  starch)  and  lipase  (split- 
ting butyrin).  Most,  but  not  all,  tumors  coagulated  milk  and  liquefied 
casein,  and  also  liquefied  gelatin  (rennin,  proteases).  Peroxidase  was 
nearly  always,  and  catalase  always,  present.  Digestion  of  fibrin,  co- 
agulated serum,  and  coagulated  egg  albumen  could  not  be  observed. 
Practically  all  tumors  split  glycogen.  Tyrosinase  could  not  be  demon- 
strated. The  fact  that  early  embryonic  tissues  were  found  poor  in 
enzymes*^  speaks  against  the  common  assumption  that  tumors  repre- 
sent strictly  an  embryonic  formation,  but  Long^^  found  that  xanthine- 
oxidase,  which  in  normal  development  does  not  appear  until  late  in 
fetal  life,  was  absent  from  primary  carcinomas  of  sheep  livers,  al- 
though normal  adult  sheep  liver  tissue  is  rich  in  this  enzyme. 

MacFadyen  and  Harden^^  studied  the  juices  obtained  by  grinding 
up  tumor  cells  made  brittle  by  liquid  air,  and  found  by  direct  methods 
(chiefly  in  breast  cancers)  invertase,  maltase,  amylase,  proteases 
acting  in  both  acid  and  alkaline  solutions,  catalase,  oxidase,  with  per- 
haps traces  of  lipase  and  peroxidase,  but  no  lactase. 

Tumors  arising  from  the  gastric  mucosa,  according  to  Waring,^" 
contain  both  pepsin  and  rennin;  those  from  the  pancreas,  both  pri- 
mary and  secondary  growths,  contain  trypsin,  steapsin,  amylase,  and 
rennin. 

(5)  Internal  Secretion. — If  tumors  are  derived  from  an  organ 
with  an  important  internal  secretion,  the  tumor  cells  in  many  cases 
produce  the  same  internal  secretion,  which  seems  to  have  the  same 
functional  properties  as  the  normally  produced  secretion.  Thus  a 
metastatic  growth  from  a  thyroid  tumor  has  been  said  to  functionate 
in  place  of  the  resected  gland;  Gierke^^  found  in  about  20  grams  of 

85  Ellinger,  Munch,  med.  Woch.,  1914  (61),  2336. 

8«  Jour.  Med.  Research,  1903  (9),  356. 

"  Ibid.,  1905  (13),  543. 

88  Jour.  Exper.  Med.,  1913  (18),  512. 

8'  Lancet,  1903  (ii),  224. 

^o  Jour.  Anat.  and  Physiol.,  1894  (28),  142. 

"  Hofmeister's  Beitr.,  1902  (3),  286. 


508  THE  CHEMISTRY  OF  TUMORS 

material  from  metastatic  thyroid  tissue  in  the  vertebral  column  about 
5  mg.  of  iodin,  which  was  a  trifle  larger  proportion  than  was  present 
in  the  thyroid  itself.  Carlson  and  WoelfeP^  found  much  iodin  in 
the  metastases  of  a  thyroid  carcinoma  of  a  dog,  while  in  another  dog 
whose  cancerous  thyroid  contained  no  iodin  the  secondary  tumors 
were  also  devoid  of  this  element.  I  have  also  analyzed  metastases 
from  a  carcinoma  of  the  thyroid  which  contained  no  demonstrable 
iodin,  despite  the  presence  of  colloid.  Marine  and  Johnson^^  found 
that  in  two  cases  of  cancer  of  the  thyroid  in  man,  and  one  in  the  dog, 
the  cancer  tissue  showed  no  ability  to  retain  iodin  given  by  mouth,  in 
contrast  to  normal  thyroid  and  simple  adenomas.  Meyer-Hiirlimann 
and  Oswald^'*  have  described  a  remarkable  case  of  cystic  carcinoma  of 
the  thyroid,  from  which  in  six  weeks  2840  c.c.  of  secretion  was  obtained 
by  puncture.  It  contained  0.077  mg.  iodin  per  10  c.c.  (the  patient 
having  previously  been  given  Kl)  as  compared  with  normal  thyroid 
'which  contains  0.4  to  4  mg.  per  10  gm.  It  contained  both  globuhn  and 
albumin,  the  former  corresponding  to  true  thyroglobulin,  even  to  in- 
creasing vagus  irritability  experimentally.  The  "adenomatous" 
nodules  of  the  thyroid  often  show  evidence  of  active  secretion,  Goetsch^^ 
having  found  their  cells  rich  in  mitochondria,  while  Graham'^*^  found 
that  they  take  up  iodin  and  metabolize  it  so  that  the  adenomatous 
tissue  produces  the  typical  thyroid  effect  on  the  development  of  tad- 
poles. Adrenal  cancers  do  not  usually  cause  Addison's  disease,  per- 
haps because  they  functionate  in  the  place  of  the  destroyed  gland 
(Lubarsch). 

In  the  characteristic  production  of  cachexia,  often  apparently  out 
of  all  proportion  to  the  amount  of  tumor  tissue,  there  would  seem  to 
be  evidence  that  a  peculiar  and  abnormal  product  of  metabohsm  is 
formed  by  cancer-cells,  and  extracts  from  cancers  have  been  found 
toxic  for  protozoa."  As  yet,  however,  it  has  been  impossible  to  demon- 
strate any  characteristic  toxic  substance  in  cancers.^**  Girard- 
Mangin^^  claims  that  malignant  tumors  contain  colloidal  poisonous 
substances  in  proportion  to  their  softness,  extracts  causing  paralysis 
and  fall  of  blood  pressure;  but  others  have  failed  to  substantiate  this.^ 
Because  of  the  constant  disintegration  of  the  tumor  tissues,  products 
of  autolysis  are  formed,  and  undoubtedly  enter  the  circulation  in 
small  quantities;  possibly  they  are  a  factor  in  the  systemic  mani- 
festations of  malignant  growths,  analogous  to  the  action  of  cleavage 

"  Amer.  Jour.  Physiol,  1910  (26),  32. 

"3  Arch.  Int.  Med.,  1913  (11),  288. 

»*  Korr.-Bl.  Schweizer  Aerzte,  1913  (43),  1468. 

»»  Bull.  Johns  Hopkins  Hosp.,  1916  (27),  129. 

"  Jour.  Exp.  Med.,  1916  (24),  345. 

"Woodruff  and  Underhill,  Jour.  Biol.  Chcin.,  1913  (15),  401;  Calkins.  Jour. 
Cancer  Res.,  1916  (1),  205  and  399. 

"*  See  Blumenthal,  Fcstschr.  f.  Salkowski,  Berlin,  1904;  Ilanseinann,  Zrit. 
Krebsforsch.,  1906  (4),  565. 

»»  Presse  M6d.,  1906,  p.  17)9;  Compt.  Rend.  Soc.  Biol.,  1909  (67),  117. 
1  See  Bruschettini  and  Barlocco,  Cent.  f.  Bakt.,  1907  (43),  664. 


I 


HEMOLYSIS  IN  CANCER  509 

products  of  foreign  proteins  which  may  produce  "protein  fever" 
and  other  toxic  effects.  It  has  often  been  observed  that  when  ex- 
tensive necrosis  is  produced  in  experimental  tumors  in  rats  and  mice 
the  animals  may  show  profound  toxemia,  presumably  because  of 
absorption  of  autolytic  products. 

Since  all  normal  tissue-cells  produce  substances  through  their  me- 
tabolism that  enter  the  circulation,  it  is  quite  certain  that  tumor-cells 
do  likewise,  and  it  is  highly  probable  that  the  presence  of  abnormal 
quantities  of  such  products,  even  if  they  are  of  quite  normal  compo- 
sition, may  cause  disturbances  in  the  body.  As  yet,  however,  no  such 
substances,  either  normal  or  abnormal,  have  been  isolated,  nor  has 
their  presence  been  demonstrated.  Numerous  isolated  observations 
of  ptomains  or  similar  substances  in  the  urine  of  cancer  patients  may 
be  found  in  the  literature,-  but  their  importance  is  extremely  question- 
able. A  large  proportion  of  cases  of  malignant  tumors  exhibit  renal 
injury  (Kast  and  Killian)^  but  whether  from  products  of  the  tumor  o? 
from  bacterial  infection  has  not  been  determined. 

Hemolytic  Substances. — -A  number  of  observers  have  described  the 
finding  of  hemolytic  substances  in  cancer  extracts.  Bard^  observed 
that  in  hemorrhagic  carcinomatous  exudates  in  serous  cavities  the 
blood  is  rapidly  hemolyzed,  which  is  not  the  case  in  exudates  from 
other  causes,  but  this  was  not  corroborated  by  Weil.^  Kullmann^ 
found  that  extracts  of  carcinomas  contain  hemolytic  substances  acting 
energetically  both  in  the  body  and  in  vitro;  these  are  soluble  in  alcohol 
and  in  water,  are  not  complex  in  composition,  are  not  specific  for  hu- 
man corpuscles,  but  are  toxic  for  all  varieties  of  corpuscles.  Micheli 
and  Donati*"'  likewise  found  hemolytic  substances  in  8  of  15  tumors, 
of  which  5  acted  on  all  varieties  of  corpuscles,  and  3  acted  on  only 
certain  varieties;  they  regard  the  hemolytic  substances  as  the  products 
of  autolj^sis  in  the  tumors.  WeiF  also  found  the  hemolytic  property 
of  tumor  extracts  to  vary  with  the  amount  of  necrosis,  and  to  depend 
on  dialyzable  hemolytic  substances  distinct  from  the  hemolysins 
of  normal  tissues.  It  is  well  known  that  among  the  products  of 
autolysis  of  normal  tissues  are  hemolytic  substances.  Whether  the 
severe  anemia  frequently  present  in  carcinoma  is  due,  either  largely 
or  in  part,  to  these  products  of  autolj^sis  is  unknown,  but  it  is  very 
probable  that  they  have  some  effect. 

Hemolysis  in  Cancer.^ — The  blood  serum  of  cancer  patients  has 
often  a  hemolytic  action  on  the  corpuscles  of  normal  persons  (Crile), 
but  this  property  is  quite  inconstant,  being  present  in  67  per  cent,  of  a 
series  of  472  cancer  cases  collected  by  Krida,  while   15  per  cent,  of 

2  See  KuUmann,  Zeit.  klin.  Med.,  1904  (53),  293. 

3  Proc.  See.  Exp.  Biol.  Med.,  1919  (16),  141. 
*  La  Semaine  M6d.,  1901  (21),  201. 

5  Jour.  Med.  Res.,  1910  (23),  86. 

6  Riforma  Med.,  1903  (19),  1037. 

^  Jour.  Med.  Res.,  1907  (16),  287. 


510  THE  CHEMISTRY  OF  TUMORS 

cases  of  other  diseases  and  2.6  per  cent,  of  normal  persons  showed 
hemolytic  activity  of  the  serum. ^  Elsberg  found  that  normal  corpus- 
cles injected  subcutaneously  into  cancer  patients  are  hemolyzed,  but 
Gorham  and  Lisser  found  this  reaction  positive  in  but  60  per  cent,  of 
their  cases,  the  subcutaneous  hemolysis  not  corresponding  at  all  to 
the  hemolytic  activity  of  the  patient's  serum  in  the  test  tube.  The 
stomach  contents  in  cancer  of  the  stomach,  when  ulcerated,  are  hemo- 
lytic (Grafe  and  Rohmer).^  The  red  corpuscles  of  cancer  patients  are 
said  to  have  usually  a  greater  resistance  to  hemolysis  by  cobra  venom 
than  normal  corpuscles,  but  this  is  not  characteristic,  there  being  simi- 
lar alterations  in  other  diseases.^*'  The  reputed  power  of  the  serum 
in  cancer  to  protect  corpuscles  from  hemolysis  by  oleic  and  lactic  acid 
could  not  be  demonstrated  by  Sweek  and  Fleisher.^^ 

An  extensive  review  of  the  literature  and  methods  led  Cohnreich^^ 
to  the  conclusion  that  resistance  of  erj^throcytes  to  hypotonic  solutions 
and  to  poisons  vary  independently  of  one  another.  He  has  devised  an 
improved  method  for  testing  resistance  to  hypotonic  solutions,  which 
seems  to  vary  directly  with  the  amount  of  stroma  and  PO4  content, 
and  finds  that  determinations  of  maximum  and  minimum  resistance  are 
of  little  value,  as  *"hese  concern  only  a  small  part  of  the  corpuscles; 
he  therefore  determines  the  "plurimum"  resistance,  involving  most  of 
the  corpuscles.  The  most  significant  results  were  obtained  in  cancer 
of  the  alimentary  tract,  in  which  an  increased  resistance  was  always 
demonstrable.  Farmachidis'^  finds  the  cobra  venom  resistance  more 
specific  for  cancer  than  do  most  other  investigators. 

(6)  Metabolism  in  Cancer. — There  are  numerous  observations 
indicating  that  cancer  cachexia  is  in  no  way  different  from  the  cachexia 
of  other  conditions.  The  behavior  of  the  nitrogen  metabolism  seems 
to  be  quite  the  same  as  in  tuberculosis  and  other  wasting  diseases. 
There  is  the  same  excessive  elimination  of  aromatic  substances  (phenol,  ^* 
indican)  and  oxyacids  (Lewin,^^  BlumenthaP^),  which  Lewin  con- 
siders to  arise  from  the  abnormal  metabolism  of  proteins,  and  not  from 
putrefactive  decomposition  in  the  tumor  or  in  the  intestines.  In  rats 
with  sarcoma,  increased  excretion  of  uric  acid  and  creatin  has  been  ob- 
served. ^^  There  is  also  the  same  excessive  elimination  of  mineral 
salts  that  is  observed  in  pulmonary  tuberculosis,  and  termed  "demin- 

*  Literature  bv  Gorham  and  Lisser,  Amer.  Jour.  Med.  Sci.,  1912  (144),  103. 
9  Deut.  Arch.'klin.  Med.,  1908  (94),  239. 

•"  Kraus,  Ranzi  and  H.  Ehrlich,  Sitz.  Ber.  Akad.  Wien.,  1910  (119),  3;  see  also 
Grunbaum,  Jour.  Path,  and  Bact.,  1912  (17),  82. 

"  Jour.  Med.  Res.,  1913  (27),  383. 

"  FoHa  Hematol.,  1913  (16),  307,  full  bibliography. 

"  Gaz.  degli  Osped.,  1915  (36),  689. 

^*  Somewhat  higher  than  average  figures  for  phenol  in  the  blood  were  found  in 
sarcoma  cases  by  Theis  and  Benedict  (.lour.  Biol.  Chcm.,  1918  (36),  99). 

1^  Deut.  med.  Woch.,  1905  (31),  218. 

i«  Festsclir.  f.  Salkowski,  Berlin,  1904. 

"  Ordway,  Jour.  Med.  Res.,  1913  (23),  301. 


METABOLISM  IN  CANCER  511 

eralization"  b}'  Robin,''*  but  no  alteration  in  the  excretion  of  chlo- 
rides.'^ As  in  other  cachexias,  the  creatin  content  of  the  muscles 
is  decreased.'"  FraenkeP'  finds  evidence  that  there  may  be  some  diffi- 
culty in  tryptoj)hano  inc^tabolisni  in  tumors  and  in  tumor  patients, 
especially  marked  with  melanotic  tumors.  Extensive  respiratory 
studies  by  Wallersteiner-^  showed  enormous  variations  in  the  amount  of 
heat  production  in  different  cases,  in  about  10  per  cent,  of  which  figures 
as  hip;h  as  those  of  severe  fevers  or  exophthalmic  goiter  were  obtained 
repeatedly;  most  of  the  cases  showed  high  normal  figures.  Nitrogen 
loss  did  not  ordinarily  occur  if  the  calorimetric  findings  were  considered 
in  the  calculations;  nitrogen  equilibrium  was  maintained  if  sufficient 
nourishment  was  obtained  and  utilized.  In  general,  metabolism  in 
cancer  resembles  that  of  fever,  and  warrants  the  assumption  of  a  toxic 
stimulation  of  tissue  destruction.  It  is  entirely  possible  that  the  pro- 
ducts of  cancer  protein  destruction  are  responsible  for  this  toxicogenic 
metabolic  abnormality,  since  Vaughan  has  demonstrated  that  the 
effects  of  bacteria  and  foreign  proteins  are  quite  the  same  in  their 
pyretic  and  toxic  action. 

Salkowski  demonstrated  that  the  amount  of  colloidal  nitrogenous 
material,  precipitated  from  the  urine  by  strong  alcohol,  is  increased  in 
cancer.  Numerous  observers  have  corroborated  this,  but  find  that  a 
similar  conchtion  obtains  in  other  cachectic  diseases,  although  in  cancer 
the  amount  of  colloidal  nitrogen  seldom  is  as  low  as  normal  unless  the 
tumor  is  removed. ^^  Much  of  this  colloidal  nitrogen  seems  to  be  in  the 
form  of  "oxy-proteic  acid"  (Salomen  and  Saxl),-'*  which  is  a  mixture 
of  incompletely  oxidized  polypeptids,  containing  much  unoxidized  sul- 
phur." The  proportion  of  neutral  sulphur  in  the  total  sulphur  in  the 
urine  seems  to  be  increased  in  cancer  (Weiss),  but  not  so  constantly 
or  characteristically  as  to  be  of  great  diagnostic  value."  Much  clinical 
investigation  has  been  made  of  these  urinary  changes,  which  has  gen- 
erally substantiated  the  fact  that  there  usually  is  more  increase  in 
colloidal  nitrogen  and  ethereal  sulphate  in  the  urine  of  cancer  than 
in  other  diseases,  but  that  in  no  sense  are  these  changes  specific  for 
cancer,  and  the  fundamental  metabolic  disturbances  responsible  have 

^^  Quoted  by  Lewin,  he.  cit.^^  Clowes  et  al.  (5th  Ann.  Rep.,  X.  Y.  State  Dept. 
of  Health,  1903-4)  report  observing  a  slight  chloride  retention  in  cancer  patients, 
and  review  the  literature  of  metabolism  in  cancer. 

'8  Robin,  Compt.  Rend.  Acad.  Sci.,  1913  (156),  1262. 

20  Chisholm,  Biochem.  Jour.,  1912  (6),  243. 

='  Wien.  klin.  Woch.,  1912  (25),  1041. 

22  Deut.  Arch.  klin.  Med.,  1914  (116),  145. 

23  See  Mancini,  Deut.  Arch.  klin.  Med.,  1911  (103),  288;  Semenow,  Foha  Urol., 
1912  (7),  215;  de  Bloeme  et  al,  Biochem.  Zeit.,  1914  (65),  345. 

2*  Wien.  klin.  Woch.,  1911  (24),  449. 

25  Killian  reports  finding  in  the  blood  two  to  three  times  the  normal  amount  of 
nonprotein  sulphur  while  the  total  sulphates  remain  normal.  ("Cancer:  Its 
Nature,  Causes,  Diagnosis  and  Treatment."     By  R.  H.  Greene,  New  York,  1918.) 

2«  Stadtmijller  and  Rosenbloom,  Arch.  Int.  Med.,  1913  (12),  276;  Interstate 
Med.  Jour.,  1916  (23),  No.  2;  bibliographv.  Kahn,  Jour.  Cancer  Res.,  1917  (2), 
379. 


512  THE  CHEMISTRY  OF  TUMORS 

not  been  ascertained.^^  They  seem  more  indicative  of  the  excessive 
catabohsm  of  cachexia  than  of  cancer  tissue  itself.  SaxP*  has  as- 
cribed part  of  the  increased  sulphur  elimination  to  abnormal  excre- 
tion of  sulphocyanid,  and  as  small  doses  of  sulphocyanides  lead  to 
increased  oxyproteic  acid  in  the  urine  he  suggests  that  in  cancer  there 
is  a  specific  disturbance  in  sulphocyanid  metabolism,  an  hypothesis 
that  awaits  confirmation.  Of  similar  status  is  the  excessive  excretion 
of  glycuronic  acid  described  by  Roger. ^^ 

Israel,  and  also  Engelmann,  have  reported  the  occurrence  of  a 
marked  increase  in  the  lowering  of  the  freezing-point  of  the  blood  in 
carcinoma  (as  low  as  —0.60°  to  —0.63°,  the  normal  being  —0.56°), 
which  they  attributed  to  the  presence  of  excessive  products  of  protein 
decomposition  in  the  blood.  Engel,^"  however,  found  no  such  in- 
creased lowering  of  the  freezing-point  in  his  cases,  and  questions  the 
significance  of  the  results  of  Israel  and  Engelmann.  There  may  be  a 
dietary  increase  in  the  blood  sugar  in  cancer, ^^  which  rises  more  rapidly 
and  remains  high  longer  than  normal.^-  The  total  protein  of  the  blood 
is  low,  with  some  increase  in  the  proportion  of  globulin  as  is  usual  in 
cachexia. ^^  According  to  Moore  and  Wilson^''  the  acid-neutralizing 
power  of  the  blood  ("alkalinity")  is  increased  in  cancer;  this  is  prob- 
ably related  to  if  not  the  cause  of  the  decreased  HCl  content  of  the 
gastric  juice,  which  occurs  whether  the  cancer  is  in  the  stomach  or  not. 
As  this  alkalinity  is  not  associated  with  an  increase  in  the  inorganic 
bases  of  the  blood,  it  may  be  that  the  proteins  have  an  increased 
basicity.  Although  numerous  other  observers  describe  a  decreased 
alkalinity  as  in  other  cachectic  conditions, ^^  Menten,^^  making  direct 
H-ion  measurements,  found  an  increase  in  alkalinity  in  the  serum  of 
nearly  all  cases  of  carcinoma  and  sarcoma.  The  blood  in  cancer 
contains  less  calcium  than  normal  which  results  in  a  tendency  to 
osteoporosis"  and  to  deposition  of  calcium  in  the  kidney  epithelium;^'* 
there  is  an  increase  in  the  potassium  of  both  the  blood  and  tissues. ^^ 
Blood  analyses  in  189  cases  of  cancer,  by  Theis  and  Stone, ^^  gave  usu- 
ally low  figures  for  non-protein  and  urea  nitrogen,  but  with  amino-N 

"  See  Goodridge  and  Kahn,  Biochem.  Bull.,  1915  (4),  118;  Damask,  Wien.  klin. 
Woch.,  1915  (28),  499;  Sassa,  Biochem.  Zeit.,  1914  (64),  195. 

28  Biochem.  Zeit.,  1913  (55),  224. 

=»Bull.  Hoc.  Med.  Hop.,  Paris,  1915  (31),  499. 

»"  Berl.  klin.  Woch.,  1904  (41),  828. 

51  Williams  and  Humphreys,  Arch.  Int.  Med.,  1919  (23),  537. 

'-  Rohdenburg,  Bernard  and  Krehbiel,  Jour.  Amcr.  Med.  Assoc,  1919  (72), 
1528. 

''  Loebner,  Deut.  Arch.  klin.  Med.,  1918  (127),  397. 

5'  Biochem.  Jour.,  1906  (1),  297:  Watson,  Jour.  Path,  and  Bact.,  1909  (13), 
429;  Sturrock,  Brit.  Med.  Jour.,  1913  (2),  780. 

»^  See  Traube,  Int.  Zeit.  Physik.-Chem.  Biol.,  1914  (1),  389. 

3"  Jour.  Cancer  Res.,  1917  (2),  179. 

"  Goldzieher,  Verb.  Deut.  Path.  Ges.,  1912  (15),  283. 

"8  M.  B.  Schmidt.,  Verb.  Deut.  Path.  Ges.,  1913  (16),  329. 

"  Mottram,  .\rch.  Middlesex  Hosp.,  1910  (19),  40. 

*"  Jour.  Cancer  Res.,  1919  (4),  349. 


DIET  AND  TUMOR  GROWTH  513 

slightly  above  normal;  uric  acid  and  sugar  were  within  normal  limits. 
Cholesterol,  fatty  acids  and  total  fats  are  generally  increased  in  the 
blood  in  malignancy.'"" 

(7)  Diet  and  Tumor  Growth. — In  general,  any  condition  that 
decreases  the  nutrition  of  tlic  body  as  a  whole,  or  of  the  tissue  in  which 
a  tumor  is  located,  decreases  the  rate  of  growth  of  the  tumor,  in  which 
respect  neoplasms  exhibit  quite  the  opposite  behavior  to  infectious 
processes.  Thus,  the  older  the  individual  the  more  slowly  the  tumor 
usually  grows;  ligation  of  the  lingual  artery  retards  the  growth  of 
cancer  of  the  tongue;  repeated  pregnancy  and  lactation  delay  the 
progress  of  cancer  in  mice,"*^  suggesting  that  tumor  cells  have  a  greater 
avidity  for  nutritive  elements  in  the  blood  than  have  ordinary  somatic 
cells,  but  less  than  the  cells  of  the  fetus  or  of  the  active  mammary  gland. 
Numerous  attempts  have  been  made  to  determine  the  relation  of  tumor 
growth  to  specific  dietary  deficiencies.  Sweet,  Corson- White  and 
Saxon"*-  found  that  rats  kept  upon  a  diet  deficient  in  specific  amino- 
acids  (lysine),  so  that  body  growth  did  not  occur  although  nutrition 
was  maintained,  show  a  slower  growth  of  implanted  tumors  than  ani- 
mals on  an  adequate  diet.  Rous"*^  obtained  similar  results  with  some 
transplanted  tumors,  but  not  with  all,  nor  with  spontaneous  tumors. 
Van  Alstyne  and  Beebe*'*  found  that  rats  living  on  casein  and  lard 
showed  much  less  growth  of  inoculated  tumors  than  when  lactose  was 
added  to  the  diet.  Robertson  and  Burnett"*^  observed  that  the  addi- 
tion of  cholesterol  to  the  diet  increases  the  rate  of  growth  and  the  de- 
velopment of  metastases  in  inoculated  rat  tumors,  which  has  been 
corroborated  by  others.  This  accelerative  action  depends  on  the  hy- 
droxyl  radical,  although  other  hydroxy-benzol  derivatives  do  not  have 
this  effect."*"  The  growth-promoting  principle  of  the  hypophysis, 
tetheUn,  is  also  said  to  stimulate  cancer  growth.  Funk"*^  found 
greater  growth  of  inoculated  sarcoma  in  fowls  given  normal  diets  than 
in  those  fed  pohshed  rice.  Benedict  and  Rahe'*^  supplied  vitamines 
by  adding  to  an  otherwise  inadequate  diet,  just  enough  yeast  to  keep 
the  rats  in  fair  condition,  and  found  that  inoculated  tumors  grew, 
although  extremely  slowly,  even  when  the  animal  itself  could  not  grow. 
Evidently  tumor  cells  cannot  manufacture  substances  essential  for 
growth  i.  e.  vitamines.  Corson-White*^  states  that,  generally, 
vitamine-rich  diets  favor  tumor  growth,  especially  if  there  is  also  an 
abundance  of  cholesterol.  Fraenkel,^°  however,  observed  no  stimulat- 
ing effect  from  rice  polishings  or  yeast  extracts. 

"»  Ee  Niord  et  al.,  Arch.  Int.  Med.,  1920  (25),  32. 

"^  See  Maud  Slve,  Jour.  Cancer  Res.,  1919  (4),  2.5. 

'-  Jour.  Biol.  Chem.,  191.3  (15),  LSI ;  1915  (21),  311. 

«  Jour.  Exp.  Med.,  1914  (20),  433. 

"  Jour.  Med.  Res.,  1913  (24),  217. 

**  Jour.  Exp.  Med.,  1913  (17),  344. 

^«  Jour.  Cancer  Res.,  1918  (3),  75. 

*'  Zeit.  phvsiol.  Chem.,  1913  (88),  352. 

^8  Jour.  Cancer  Res.,  1917  (2).  1.59. 

"  Penn.  Med.  Jour.,  1919  (22),  348. 

*"  Wien.  klin.  Woch.,  1916  (29),  483. 

33 


514  THE  CHEMISTRY  OF  TUMORS 

(8)  Immunity  Reactions  in  Cancer. — The  fact  that  a  certain  degree 
of  specific  immunity  can  be  developed  against  normal  tissue  cells  (see 
Cytotoxins,  Chap,  x),  has  encouraged  study  of  the  possibility  of  se- 
curing immune  antibodies  which  might  be  specific  for  cancer,  and  has 
led  to  much  research  on  this  subject, ^^  with  results  as  yet  of  httle 
value.  There  is  no  doubt  that  the  bodj^  has  distinct  powers  to 
inhibit  to  a  greater  or  less  degree  the  growth  of  tumors,  and  to 
destroy  many  of  the  cells  which  escape  from  cancers  into  the  lymph 
and  blood,  ^2  while  in  experimental  animals  inoculated  tumors  are 
in  most  instances  unable  to  grow,  and  they  may,  after  growth  has 
once  begun,  recede  or  even  disappear.  Furthermore,  animals  may 
be  made  immune  to  tumors  to  which  they  would  otherwise  be  susceptible . 
Many  schemes  of  immunization  of  patients  by  injection  of  extracts 
or  autolysates  made  from  their  own  tumors,  or  similar  tumors  of  others, 
have  been  tried,  ^^  but  in  the  hands  of  competent  and  critical  observers 
the  results  seem  to  have  been  practically  negative.  ^^  It  is  not  always 
kept  in  mind  that  inoculated  cancers  in  rats  and  mice  represent  an 
artificial  condition  behaving  very  differently  from  spontaneous  tumors. 

There  is  no  lack  of  evidence  that  cancers  do  produce,  in  greater  or 
less  amounts,  various  antibodies  of  some  degree  of  specificit}'  for  can- 
cer, which  must  be  interpreted  as  evidence  that  cancer  proteins  are  in 
some  respects  different  from  the  normal  proteins  of  the  host;  however, 
the  amount  and  specificity  of  these  antiboches  seem  to  be  low," 
and,  in  many  observations,  they  have  failed  to  be  demonstrated.  In- 
deed, Coca  in  his  review  states  unqualifiedlj^,  "The  usual  biological 
tests  of  complement  deviation  and  specific  precipitation  fail  to  show 
the  hypothetical  antibodies,  though  a  distinct  cytotoxic  influence  can 
be  demonstrated  in  the  plasma  of  animals  of  foreign  species  that  have 
been  actively  immunized  against  a  tumor."  His  own  experiments 
failed  to  demonstrate  specific  complement-fixation  antibodies  in 
patients  injected  with  extracts  of  their  own  tumors.  Lewin^'^  also 
fails  to  find  conclusive  evidence  of  the  demonstration  of  specific  anti- 
bodies in  cancer,  yet  accepts  the  immunity  which  is  produced  by  in- 
jections of  virulent  cancer  material  as  an  active  immunity  dependent 
upon  cancer  antibodies.  It  may,  however,  depend  on  a  stimulation 
of  the  local  cellular  reactions  that  inhibits  cancer  growth.  ^^  Pfeiffer^'* 
claims  to  find  specific  anaphylactic  antibodies  in  the  blood  of  cancer 
patients,  but  this  has  not  been  confirmed  by  several  other  observers.  ^'■' 

"  Literature  by  Coca,  Zeit.  Immunitat.,  1912  (13),  525;  Kraus  et  al.,  Wien 
klin.  Woch.,  1911  (24),  1003. 

"  Reviewed  by  Wells,  Jour.  Amer.  Med.  Assoc,  1909  (52),  1731. 

"  Review  by  Fichera,  Jour.  Cancer  Res.,  1918  (3),  303. 

"See  Blumenthal,  Zeit.  Krebsforsch.,  1914  (14),  491;  Bauer,  Latzel  and 
Wessely,  Zeit.  klin.  Med.,  1915  (81),  420. 

"  See  Morgenroth  and  Bieling,  Biochem.  Zeit.,  1915  (68),  85. 

^«  Folia  Serologica,  1911  (7),  1013;  literature. 

"  Tyzzer,  Jour.  Cancer.  Res.,  1910  (1),  125. 

"  Wien  klin.  Woch.,  1909  (22),  989;  Zeit.  Immunitiit.,  1910  (4),  455. 

"  See  Weil,  Jour.  Exp.  Med.,  Oct.,  1913,  (18),  390. 


IMMUNITY  REACTIONS  IN  CANCER  515 

V.  Dungern^"  has  described  positive  complement  fixation  reactions, 
partially  specific  for  cancer  and  benign  tumors,  by  using  alcoholic 
extracts  of  the  tumors  or  acetone  extracts  of  human  erythrocytes  as 
antigen,  but  he  interprets  these  reactions  as  not  due  to  specific  anti- 
bodies, but  to  abnormal  products  of  metabolism.''^  The  complement 
content  of  the  blood  is  said  to  be  slightly  increased  in  cancer  (Engel),^- 
but  there  is  nothing  characteristic  about  this.  Ascoli  and  Izar*^ 
have  applied  the  mciostagmin  test  {q.  v.)  and  state  that  this  gives 
very  positive  results  in  determining  the  existence  of  cancer,  their 
work  having  been  corroborated  by  many  but  not  by  all  of  those  who 
have  repeated  it.^^  Burmeister'"'^  could  obtain  no  reliable  results  with 
the  epiphanin  reaction. 

Freund  and  Kaminer'"'  have  found  that  the  serum  of  cancer  pa- 
tients is  unable  to  dissolve  cancer  cells,  as  normal  serum  does,  and 
even  protects  them  against  the  lytic  power  of  normal  serum.  The 
lysis  is  ascribed  to  a  non-nitrogenous  fatty  acid,  while  the  protective 
agent  of  cancer  serum  is  said  to  be  a  "nucleo-globulin"  which  is  in- 
creased in  the  serum  in  cancer.  They  also  find  that  cancer  extracts 
give  a  specific  turbidity  or  precipitation  with  cancer  serum,  which 
is  attributed  to  a  carbohydrate  content  of  the  extract.  According 
to  Kraus  and  v.  Graff"  the  serum  of  full  term,  pregnant  women, 
and  normal  umbilical  cord  serum,  behave  like  serum  from  cancer 
patients.  In  support  of  Freund  and  Kaminer's  observation  is  the 
experiment  of  Neuberg^^  who  found  that  cancer  cells  plus  normal 
serum  underwent  digestion  more  rapidly  than  cancer  cells  plus  cancer 
serum,  as  measured  by  the  incoagulable  nitrogen.  A  critical  test  of 
many  recommended  methods  of  serum  diagnosis  of  cancer  by  Hal- 
pern^^  gave  disappointing  results.  With  the  von  Dungern  technic  he 
obtained  80  per  cent,  of  positive  results,  with  the  meiostagmin  reaction 
85  per  cent.,  but  with  the  Abderhalden  method  but  30  per  cent.  The 
other  methods  he  finds  of  little  value.  The  testimony  concerning  the 
specificity  of  the  Abderhalden  reaction  (q.  v.)  in  cancer  is  so  conflicting 
that  it  seems  unprofitable  to  discuss  it,  the  results  varying  from  such 

«»  Miinch.  med.  Woch.,  1912  (59),  65,1093  and  285-4;  also  Rosenberg,  Deut.med. 
Woch.,  1912  (38),  1225. 

^^  Farmachidis  (Riforma  med.,  1918  (34),  382)  states  that  onh^  with  maUgnant 
disease  occurs  the  activation  by  cobra  venom  of  the  hemolytic  action  of  the  serum 
in  the  complement  fixation  reaction. 

«-  Deut.  med.  Woch.,  1910  (36),  986.  Not  corroborated  by  Ordway  and  Kel- 
lert,  Jour.  Med.  Research,  1913  (28),  287. 

"  Munch,  med.  Woch.,  1910  (57),  2129;  Biochem.  Zeit.,  1910  (29),  13. 

"  See  Rosenberg,  Deut.  med.  Woch.,  1913  (39),  926;  Wissung,  Berl.  klin.  Woch., 
1915  (52),  998.     Roffo,  Revista  Inst.  Bact.,  Buenos  Aires,  1917  (1),  53. 

"  See  Burmeister,  Jour.  Inf.  Dis.,  1913  (12),  459;  Bruggemann,  Mitt.  Grenz. 
:Med.  u.  Chir.,  1913  (25),  877. 

««  Biochem.  Zeit.,  1912  (46),  470;  Wicn.  klin.  Woch.,  1911  (24),  1759;  1913 
(26),  2108. 

"  Wien.  klin.  Woch.,  1911  (24),  191. 

«s  Biochem.  Zeit.,  1910  (26),  344. 

"  Mitt.  Grenz.  Med.  Chir.,  1913  (27).  370.  See  also  Mioni,  Tum6ri,  1914  (3), 
697. 


516  THE  CHEMISTRY  OF  TUMORS 

as  those  cited  by  Halpern  above,  to  100  per  cent,  correct  reactions  de- 
scribed by  others. ''''  Coca'^^  obtained  entirely  unsatisfactory  results 
with  both  the  von  Dungern  complement  fixation  test  and  the  Freund- 
Kaminer  reaction. 

Many  observations  have  been  made  on  the  antitryptic  activity  of 
the  blood  in  cancer  (see  Chap,  iii)  which  has  usually  shown  an  increase 
(in  all  but  about  10  per  cent,  of  the  cases) ;  but  many  other  conditions, 
especially  cachexia,  may  cause  positive  reactions.  Cancer  serum  is 
said  to  have  a  lessened  power  to  activate  pancreatic  lipase'^  when 
the  disease  is  progressive,  but  on  improvement  or  recovery  this  effect 
is  increased. 

B.  CHEMISTRY  OF  CERTAIN  SPECIFIC  TUMORS 

In  the  literature  are  to  be  found  a  few  studies  of  chemical  features 
of  some  forms  of  tumors,  which  may  be  briefly  discussed  to  advantage. 

(1)  Benign  Tumors 

(a)  Fibromas  and  Myomas. — The  few  specimens  studied  show 
but  a  small  amount  of  nucleoprotein,  as  might  be  expected  from  the 
small  amount  of  their  nuclear  material.  Because  of  the  tendency  of 
fibromas  to  undergo  retrogressive  changes,  the  amount  of  calcium  is 
likely  to  be  large.  No  studies  as  to  the  special  features  of  their  col- 
lagen, as  compared  with  normal  connective-tissue  collagen,  seem  to 
have  been  made.  Lubarsch"  found  no  glycogen  (microscopically) 
in  any  of  66  fibromas  he  examined.  Wells  and  Long^^*  found  that  in 
uterine  fibro-myomas  but  one  per  cent,  of  the  total  nitrogen  is  purine 
nitrogen,  distributed  as  guanine,  44  per  cent.;  adenine,  31  per  cent.; 
hypoxanthine,  25  per  cent.  The  relatively  large  proportion  of  pre- 
formed hypoxanthine  corresponds  with  the  abundance  of  this  purine 
free  in  normal  unstriated  muscle.  Fibromyomas  are  able  to  deami- 
dize  their  guanine  and  adenine  to  xanthine  and  hypoxanthine,  and  con- 
tain guanase  but  not  adenase.  Extracts  from  uterine  fibromyomas 
show  practically  the  same  composition  as  extracts  of  normal  uterus.''* 

A  uterine  fibroid  analyzed  by  Beebe^*^  contained  14.56  per  cent,  of 
nitrogen,  0.981  per  cent,  of  sulphur,  0.139  per  cent,  of  phosphorus, 
0.013  per  cent,  of  iron,  0.12  per  cent,  of  calcium  oxide,  0.44  per  cent, 
of  potassium,  and  1.115  per  cent,  of  sodium.  The  proportions  of  ni- 
trogen and  sulphur  are  high  as  comi)ared  with  most  tumors;  the 
phosphorus,  iron,  and  potassium  are  low,  corresponding  to  the  small 
amount  of  nucleoprotein  and  the  slow  rate  of  growth.     If  degeneration 

'"  See  de  Crinis  and  Mahncrt,  Ferine ntfrsch.,  1918  (2),  103. 

^1  Jour.  Cancer  Research,  1917  (2),  61. 

"  Shaw-Mackenzie,  Lancet,  Nov.  8,  1919. 

"  Virchow's  Arch.,  1906  (183),  188. 

7*  Zeit.  Krebsforsch.,  1913  (12),  59S. 

T"*  Winiwarter,  Arch.  f.  Gyniik.,  1913  (100),  530. 

"  Amer.  Jour.  Physiol.,  1904  (12),  167. 


BENIGN  TUMORS  517 

is  marked,  the  amount  of  calcium  is  greatly  increased.  Krawkow^^ 
found  a  trace  of  chondroitin-sulphuric  acifl  in  a  uterine  fibroid.  Lu- 
barsch  found  glycogen  occasionally  in  richly  cellular  uterine  leio- 
mj'omas,  and  in  the  vicinity  of  dcgonorating  areas;  however,  76  out 
of  85  showed  no  glycogen.  Pfannensticl''*  analyzed  the  alkaline  fluid 
of  a  cystic  fibromyoma,  which  coagulated  spontaneously;  it  contained 
sugar,  but  no  mucin  or  pseudomucin.  The  cysts  were  dilated  lymph- 
spaces,  and  the  fluid  corresponded  to  lymph  in  composition.  A  similar 
result  was  obtained  by  Oerum,^'-*  who  found  in  the  fluid  scrum- 
albumin,  serum-globuhn,  and  0.358  per  cent,  of  fibrin;  the  total  pro- 
teins constituted  6.3056  per  cent.  Sollmann^"  found  in  the  "colloid" 
of  a  cystic  degenerated  fibromyoma  both  pseudomucin  and  paramucin 
(see  "Ovarian  Cysts"),  which  differed  somewhat  from  the  same  sub- 
stances found  in  ovarian  tumors. 

The, common  occurrence  of  marked  cardiac  weakness  in  patients 
with  uterine  fibroids  has  led  to  the  suggestion  that  in  the  fibroids  some 
toxic  product  is  formed  which  acts  on  the  heart,  or  that  both  the  fibroid 
and  the  heart  defect  might  result  from  a  common  cause.  The  experi- 
mental evidence  concerning  the  relationship  is  not  convincing,  and 
there  is  much  ground  for  the  belief  that  the  heart  suffers  solely  from  the 
anemia  common  in  these  cases. ^"^  There  is  said  to  be  a  hemolytic  poi- 
son, a  lipoid  according  to  Murray, ^^  formed  in  the  degenerating 
fibroids  which  causes  local  hemolysis  and  "red  degeneration,"  and 
there  are  cases  of  acute  aseptic  degeneration  of  fibromyomas  which 
seem  to  have  caused  systemic  intoxication. 

(6)  Myxomas. — From  a  myxoma  of  the  back  Oswald*'  obtained  a 
mucin  with  the  following  elementary  composition:  C,  50.82;  H,  7.27; 
N,  12.24;  S,  1.19;  P,  0.25  per  cent.  This  differs  from  other  mammalian 
mucins  in  the  presence  of  phosphorus,  but  Oswald  does  not  consider 
this  a  contamination.  It  also  contained  12  per  cent,  of  carbohydrate, 
apparently  glucosamine. 

(c)  Chondromas,  like  normal  cartilage,  always  contain  much 
glycogen  (Lubarsch).  Morner^^  found  chondroitin-sulphm'ic  acid 
in  several  chondromas  that  he  examined,  although  Schmiedeberg  had 
failed  to  do  so. 

'^  Arch.  exp.  Path.  u.  Pharm.,  1898  (40),  195. 

"Arch.  f.  Gyn.,  1890  (38),  468. 

"  Maly's  Jahresber.,  1884  (14),  462. 

8«  Amer.  Gynecol.,  1903  (2),  232. 

81  See  Jaschke,  Mitt.  Grenz.  Med.  u.  Chir.,  1912  (15),  249;  ;McGUnn,  Surg. 
Gyn.,  Obst.,  1914  (18),  180. 

82  Jour.  Obs.  and  Gyn.,  1910  (17),  534. 
"  Zeit.  physiol.  Chem.,  1914  (92),  144. 
s^Zeit.  phvsiol.  Chem.,  1895  (20),  357. 


518 


THE  CHEMISTRY  OF  TUMORS 


(d)  Lipomas^^  have  been  studied  by  Schulz^*'  and  by  Jaeckle.^^ 
The  former  found  in  a  retroperitoneal  hpoma  75.75  per  cent,  of  fat, 
2.25  per  cent,  of  connective  tissue,  and  22  per  cent,  of  water.  Of  the 
fat,  7.31  per  cent,  was  in  the  form  of  the  free  fatty  acids  and  92.7  per 
cent,  as  neutral  fats.  The  fatty  acids  of  the  fat  consisted  of  65.57 
per  cent,  oleic  acid;  29.84  per  cent,  stearic  acid;  4.59  per  cent,  pal- 
mitic acid.  Cholesterol  was  only  qualitatively  demonstrable.  In  the 
connective  tissue  was  found  chondroitin-sulphuric  acid.  Lubarsch 
found  glycogen  in  lipomas  only  when  they  were  degenerated.  Jaeckle 
observed  the  formation  of  calcium  soaps  in  a  calcifying  lipoma,  the 
calcium  being  distributed  as  follows:  calcium  soaps,  29.5  per  cent.; 
calcium  carbonate,  28.61  per  cent.;  calcium  phosphate,  41.89  per  cent. 
The  fats  of  lipomas  he  found  practically  identical  with  those  of  the 
subcutaneous  tissues,  except  sometimes  for  a  deficiency  in  lecitliin, 
as  shown  by  the  following  figures: 

Composition  op  Fats  in — 


Subcutane- 
ous tissue 

Lipoma 

I 

Lipoma 
II. 

Lipoma 
III 

Refraction,  at  40° 

50.6 
197.3 
0.25 

63.7 

74.1 

70.9 
0.39 
0.196 

18.5 
6.2 
0.084 
0.32 

50.1 

197.7 
0.33 

59.0 

68.6 

65.7 
0.31 
0.155 

24.9 
5.1 

50.9 
197.7 
0.35 
64.0 
74.4 
71.2 
0.48 
0.24 

'0.'34 

50.5 

Saponification  number 

Reichert-Meisser  number. . .  . 
lodin  number 

195.9 
0.35 
64.1 

Olein 

74.5 

Oleic  acid 

71.3 

Acid  number 

0.67 

Free  acid 

0.34 

Palmitic  acid 

18.5 

Stearic  acid 

5.9 

Lecithin 

Cholesterol 

0.015 

Lipomas  are  able  to  hydrolyze  fats  and  esters,  their  lipase  behaving 
in  all  respects  like  the  lipase  of  normal  areolar  tissue.****  Lipoma  fat 
is  hydrolyzed  by  lipase  as  readily  as  is  normal  human  fat.  No  rea- 
son for  the  reputed  unavailability  of  lipoma  fat  for  the  metabolism 
of  the  host  could  be  found.     It  is  doubtful  if  the  fat  of  benign  lipomas 

*^  In  xanthoma  tuberosum  multiplex,  which  shows  local  deposits  composed  largely 
of  cholesterol  esters  and  contains  also  pigment  with  the  properties  of  a  lipochrome, 
the  presence  of  hyper-cholesterolemia  is  disputed.  (Roscnbloom,  Arch.  Int.  Med., 
1913  (12)  395;  Schmidt,  Dermatol.  Zeit.,  1914  (21),  i:37). 

Edsall  found  the  composition  of  the  fat  in  the  fatty  tumors  of  adiposis  dolorosa 
(Dercum's  disease)  but  little  different  from  that  of  normal  fat.  (Cjuotod  by 
Dercum  and  McCarthy,  Amer.  Jour.  Med.  Sci.,  1902  (124),  994).  Martelli 
(Tumori,  1918  (6),  1)  on  histological  grounds  states  that  in  2-3  per  cent,  of  the 
cells  the  fats  are  mi.xed  with  fatty  acids,  while  cholesterol,  phospholijiins  and 
chromolijjins  are  very  scanty;  he  attributes  the  condition  to  disturbed  lipogenesis 
from  enducrin-sympathetic  disfunction. 

8"  Pfluger's  Arch.,  1893  (55),  231. 

8^  Zeit.  physiol.  Chem.,  1902  (36),  53. 

88  Wells,  Arch.  Int.  Med.,  1912  (10),  297;  full  review. 


OVARIAN  CYSTS  519 

is  entirely  unavailable  for  metabolism,  at  least  in  all  cases,  but  in 
malignant  fatty  tumors  this  seems  to  be  true.  Hirsch*^  and  Wells 
have  studied  such  a  tumor  in  which,  despite  most  complete  exhaus- 
tion of  the  fat  from  the  normal  fat  depots,  about  two  pounds  of  fat 
and  four  and  one-half  pounds  of  protein  were  stored  up  in  the  growing 
tumor.  This  tumor  was  an  edematous  retroperitoneal  lipo-sarco?na, 
weighing  G9  pounds  (the  heaviest  solid  tumor  on  record)  and  its 
chemical  composition  is  given  below  as  compared  with  the  composition 
of  granulation  tissue  (castration  granuloma  of  swine) . 

Fibro  lipo 

sarcoma.  Granuloma, 

per  cent.  per  cent. 

Alcohol  ether  residue 6 .  53  15 .  84 

Alcohol  ether  extract 2.94  2.28 

Total  soUds 9.47  18.12 

Water 90.53  81.88 

Alcohol  ether  residue 
(per  cent,  of  solids) 

Total  protein 66.91  91.16 

Protein  sulphur 0 .  65  0 .  33 

Protein  phosphorus 0 .  44  0.31 

Total  Purine  Nitrogen 0. 13  0. 07 

Lipins  contained, 
per  cent. 

Total  nitrogen 0.17  0 .  09 

Total  sulphur 0 .  13  0 .  05 

Total  phosphorus 0.19  0 .  09 

(e)  Ovarian  cyst  contents  have  been  studied  more  than  almost 
any  other  tumor  products,  because  in  their  gelatinous  or  slimy  sub- 
stance are  contained  numerous  interesting  forms  of  proteins,  many 
of  which  are  combined  with  carbohydrates  and  related  to  the  true 
mucins.  These  substances  are  frequently  referred  to  under  the  names 
of  pseudomucin,  paralbumin,  metalbumin,  and  ovarian  "colloid,"  and 
belong  to  the  class  of  "mucoids."^°  In  view  of  the  fact  that  the  flu- 
ids in  the  Graafian  follicles  of  the  ovary  do  not  contain  these  particu- 
lar forms  of  protein,  their  presence  in  cysts  derived  from  adventitious 
structures  (Pfliiger's  epithelial  tubes)  suggests  a  specific  form  of  meta- 
bolism on  the  part  of  the  epithelium  of  these  structures. 

Serous  cysts,  formed  by  dilation  of  Graafian  follicles,  usually  are 
small  in  size,  and  the  contents  resemble  those  of  the  normal  follicles 
(Oerum),^^  consisting 'of  a  serous  fluid  with  a  specific  gravity  usually 
from  1.005  to  1.014  (occasionally  1.020  or  more),  and  containing 
1.0-4.0  per  cent,  of  solids.  Occasionally  in  these  cysts  the  contents 
become  solidified  through  absorption  of  the  water,  and  a  gelatinous  or 
glue-like  "colloid"  content  results.  Mucoids  are  never  present  (Pfan- 
nenstiel).^- 

89  Amer.  Jour.  Med.  Sci.,  1920  (159),  356. 

9"  Concerning  mucoids  see  Mann's  "Chemistry  of  the  Proteins,"  1906,  pp.  541- 
551. 

"  See  Malv's  Jahresbericht,  1884  (14),  459. 

92  Arch.  f.^Gynsek.,  1890  (38),  407  (literature). 


520  THE  CHEMISTRY  OF  TUMORS 

Proliferating  cystomas  contain  the  peculiar  characteristic  mucoid 
proteins  mentioned  above.  Usually  the  contents  are  fluid,  but  of  a 
pecuhar  slimy,  stringy  character,  due  to  the  mucoid  substance,  and 
often  opalescent  or  slightly  turbid.  The  specific  gravity  is  generally 
high — 1.015-1.030.  The  reaction  is  usually  slightly  alkaline  to  lit- 
mus, and  neutral  or  slightly  acid  to  phenolphthalein.  If  hemorrhage 
has  occurred  into  them,  the  fluid  is  discolored,  and  may  contain  blood- 
pigments  in  crystalhne  and  amorphous  forms.  Small  cysts  often 
show  a  condensation  of  the  proteins  into  a  semisolid  "colloid"  ma- 
terial, but  sometimes  their  contents  resemble  those  of  a  serous  cj^st. 
Often  masses  of  proteins  fall  out  of  solution,  forming  yellowish  floc- 
culi  or  large  deposits  half  filling  the  cysts.  As  with  all  stagnant 
fluids  of  this  type,  cholesterol  crystals  are  frequently  found.  The  char- 
acteristic proteins  are  members  of  the  class  of  pseudomucins,  wliich 
are  constantly  present  (Oerum). 

Intraligamentary  papillary  cysts  contain  a  j^ellow,  yellowish-green, 
or  brownish-green  liquid,  which  contains  little  or  no  pseudomucin; 
the  specific  gravity  is  usually  high  (1.0.32-1.036)  and  the  fluid  con- 
tains 9  to  10  per  cent,  of  solids.  The  principal  constituents  are  the 
simple  proteins  of  blood  serum  (Hammarsten). 

According  to  the  same  author,  the  rare  tubo-ovarian  cysts  contain  a 
watery  serous  fluid  with  no  pseudomucin. 

Chemistry  of  the  Mucoids  of  Ovarian  Cysts. — Pseudomucin  has  the  following 
elementary  composition:  C,  49.75;  H,  6.98;  N,  10.28;  S,  1.25;  O,  31.74  per  cent. 
(Hammarsten).  In  common  with  the  true  mucins  it  yields  a  sugar-like  reducing 
body,  which  has  been  investigated  by  numerous  chemists  (Miiller,  Panzer,  Zan- 
gerle,  Leathes,  Neuberg,  and  Heymann*').  Panzer  considers  that  this  reducing 
substance  is  in  the  form  of  a  sulphuric-acid  compound,  similar  to,  but  not  identical 
with,  chondroitin-sulphuric  acid.  Hammarsten,  however,  did  not  find  this 
substance  constantly  present.  Leathes  determined  for  the  carbohydrate  group 
the  composition  C12H23NO10,  named  it  " paramucosin,"  and  considers  it  a  reduced 
chondrosin  (which  is  the  carbohydrate  group  of  chondroitin-sulphuric  acid). 
Neuberg  and  Heymann  established,  however,  that  the  reducing  body  must  come 
from  chitosamin  (CeHuNOs),  and  do  not  consider  paramucosin  a  constant  con- 
stituent of  ovarian  mucoids.  The  amount  of  reducing  substance  varies  greatly 
in  the  mucoids  found  in  different  cysts;  in  some  the  mucoid  yields  but  about 
3  to  5  per  cent.,  in  others  as  much  as  30  to  35  per  cent.,  of  reducing  substance. 

Pseudomucin  dissolves  readily  in  weak  alkalies,  and  differs  from  true  mucin 
in  that  it  is  not  precipitated  by  acetic  acid,  and  from  the  simple  proteins  in  that 
its  solutions  are  not  coagulated  by  boiling.  With  water  a  slinn',  stringy,  semi- 
solution  is  formed,  resembling  in  appearance  the  material  found  in  ovarian  cysts. 
Leathes  distinguishes  two  forms  of  ovarian  mucoids:  One,  paramucin,  occurs  as 
a  firm,  jelly-like  substance,  which  is  converted  by  peptic  digestion  into  easily 
soluble  pseudomucin.  Ovarian  "coZ/oz'd"  probably  consists  of  a  thickened  pseudo- 
mucin, often  mixed  with  other  proteins.  Pfannenstiel*-  considers  the  "colloid" 
material  as  representing  a  modified  pseudomucin,  strongly  alkaline  and  relatively 
insoluble,  which  he  calls  "  pseudo-mucin  /i."  He  also  describes  a  very  soluble 
mucoid  found  only  in  certain  ovarian  cysts,  naming  it  "  pseudo-jriucin  y." 

The  reason  why  these  variations  in  the  pseudomucins  exist  is  not  understood; 
they  cannot  be  explained  as  due  to  variations  in  tiie  cell  type  in  the  ej'st  wall, 
although  pseudomucin  is  probably  the  result  of  true  secretion.  The  smallest 
cavities  of  ovarian  cystadenomas  contain  nearly  pure  pseudomucin,  which  presents 

•3  Hofmeister's  Beitr.,  1902  (2),  201  (literature) 


OVARIAN  CYSTS  521 

a  clear,  glassy  structure;  the  larger  the  cysts  become,  and  the  more  turbid  and 
thinner  the  fluid  is,  the  more  .'im])Ie  are  the  proteins  it  contains.  True  mucin 
is  never  present  in  ovarian  cysts.  Pseudomucin  occurs  only  in  the  glandular 
proliferating  cystomas  and  the  papillary  proliferating  cystadenomas,  in  the  former 
appearing  constantly  and  abundantly,  in  the  latter  not  constantly  and  never 
abundantly  (Pfanncnstiel).  Paralhximin  (Scherer)  is  a  mi.\ture  of  pseudomucin 
with  vari;ii)le  amounts  of  simple  proteins.  Mctnlbumin  (Schercr)  is  the  same  body 
that  is  called  pseudomucin  by  Ilamiuarsten.  Pnramucin  (MitjukotT)"*  is  a  mucoid 
differing  from  mucin  and  pseudomucin  in  reducing  Fehling's  solution  directly, 
without  having  the  carbohydrate  group  first  split  off  by  boiling  with  an  acid. 
Hydrolysis  of  paramucin  by  PregP''  showed  an  absence  of  glycine,  but  traces  of 
diamino-acids,  and  the  presence  of  leucine,  alanine,  proline,  aspartic  and  glutamic 
acids,  tryptophane  and  tyrosine. 

Substances  similar  to  pseudomucin  have  been  occasionally  found  in  cancerous 
ascitic  fluid  and  in  cystic  fibromyomas  (HoUmann);  and  they  are  abundant  as 
constituents  of  the  contents  of  the  peritoneum  in  the  condition  known  as  "pseudo- 
myxoma peritonei,"^^  when  the  material  is  in  realit.y  the  product  of 
cells  implanted  on  the  peritoneal  surface  through  the  bursting  of  an  ovarian 
cyst  (or  a  cyst  of  the  vermiform  appendix  (Frankel)).^^  The  physically  similar 
substance  found  in  pathological  synovial  membranes  by  Hammarsten  differs  in 
yielding  no  reducing  substance.  Parovarian  cysts  arising  from  the  Wolffian  body 
present  an  entirely  different  content,  which  is  a  clear,  watery  fluid,  with  specific 
gravitj'  usually  under  1.010;  the  solids  amount  to  but  1  or  2  per  cent.,  and  consist 
chiefly  of  salts  (the  ash  being  often  over  80  per  cent.),  mostly  sulphates  and  chlorides. 
They  are  usually  (or  always)  free  from  pseudomucin,  mucin,  or  other  sugar- 
containing  substances,  and  other  proteins  occur  only  in  small  amounts,  unless  the 
cyst  is  inflamed.  Apparently  mucoids  do  not  form  in  cysts  lined  by  ciliated 
epithelium  (Pfannenstiel). 

Santi^*  has  studied  the  physical  chemistry  of  ovarian  cysts,  and  finds  the  freez- 
ing point  very  near  that  of  blood,  having  no  relation  to  density,  viscosity  or  nitrogen 
content;  the  specific  electrical  conductivity  is  higher  than  that  of  blood  serum. 
The  physicochemical  properties  are  less  dependent  upon  chlorides,  and  more  on 
other  substances  (Gruner).^^ 

(/)  Dermoid  cysts  of  the  ovary  contain,  as  their  chief  and  most 
characteristic  constituent,  a  yellow  fat,  which  melts  at  34°-39°  and 
soUdifies  at  20°-25°.  Ludwig  and  Zeynek^  have  examined  over  sixty 
such  tumors,  and  found  that  the  fatty  material  constantly  contains 
two  chief  constituents:  one,  crystaHizing  out  readily,  they  believed  to 
be  cetyl  alcohol, 

(CHs  —  (CHo)  u  —  CH2OH) ; 

the  other,  remaining  as  an  oily  fluid,  seems  to  be  closely  related  to 
cholesterol,  although  not  consisting  of  one  substance  alone.  Small 
quantities  of  arachidic  acid  (Cvq,  H40O2),  as  well  as  stearic,  'palmitic 
and  myristic  acid  (C14H28O2),  existing  as  glycerides,  are  also  "pres- 
ent. Ameseder,^  however,  found  evidence  that  the  supposed  cetyl 
alcohol  is  really  eikosyl  alcohol  (C20H42O).  These  substances  are  se- 
creted by  the  glands  of  the  cutaneous  structures  of  the  cyst,  and  re- 
s'" Arch.  f.  Gynsek.,  1895  (49),  278. 

35  Zeit.  physiol.  Chem.,  1908  (58),  229. 

9«  Literature  by  Peters,  Monatschr.  f.  Geb.  u.  Gyn.,  1899  (10),  749;  Weber,  St. 
Petersb.  med.  Woch.,  1901  (26),  331. 

"  Miinch.  med.  Woch.,  1901  (48),  965. 

s^  Folia  clin.  chimica  et  microscop.,  1910  (2),  73. 

39  Biochem.  Jour.,  1907  (2),  383. 
iZeit.  phvsiol.  Chem.,  1897  (23),  40. 

2  76id.,  1907  (52),  121. 


522  THE  CHEMISTRY  OF  TUMORS 

semble  in  composition  sebaceous  material,  which  is  characterized  by 
containing  a  large  proportion  of  cholesterol  partly  combined  with  fatty 
acids.  Dermoids  sometimes  contain  masses  of  fattj'  concretions  which 
seem  not  to  depend  on  chemical  changes  but  on  the  presence  of  forma- 
tive nuclei  and  framework  of  desquamated  epithelium;  they  consist  of 
a  mixture  of  neutral  fats  and  cholesterol  esters,  with  some  free  cho- 
lesterol.^ Cholesteatomas,  in  addition  to  their  abundant  cholesterol 
content,  contain  keratin.'' 

{g)  "Butter"  Cysts." — In  the  mammary  gland  retention  cysts 
form,  filled  with  products  of  alteration  of  the  milk,  including  butyric 
acid  and  lactose  (Klotz),®  and  these  are  called  "butter  cysts"  or 
milk  cysts.  Analysis  of  the  contents  of  such  a  cyst  by  Smita^  gave 
the  following  results,  as  compared  with  human  milk: 

Cyst  contents    Human  milk 

Fat 72.97  3.90 

Casein 4.37  0.63 

Albumin 1.91  1.31 

Milk-sugar 0.88  6.04 

Ash 0.36  0.49 

Water 20.81  87.09 

Fats  consisted  of — 

Cyst  Cows'  milk 

Stearin  and  palmitin 37.0  50.0 

Olein 53.0  42.2 

Butyrin 9.0  7.8 

Occurring  independent  of  lactation  usually,  but  not  always,  are  the 
''soap  cysts,"  which  contain  chiefly  calcium  and  magnesium  soaps, 
but  also  neutral  fats,  free  fatty  acids,  and  traces  of  cholesterol 
(Freund).8 

(2)  Malignant  Tumors 

The  chief  general  features  of  the  composition  of  these  growths 
have  been  considered  in  the  discussion  of  the  chemistry  of  tumors 
in  general.  A  malignant  tumor  differs  from  a  similar  benign  tumor 
chiefly  in  having  usually  a  larger  proportion  of  the  primary  cell  con- 
stituents, and  a  smaller  proportion  of  the  secondary  constituents  and 
intercellular  substances,  since  these  are  largely  the  product  of  the 
functional  activity  of  the  cells,  which,  in  malignant  tumors,  do  not 
often  develop  sufficiently  to  functionate  extensively.  Hence  malig- 
nant tumors  usually  show  a  rather  high  proportion  of  the  characteristic 
constituents  of  nucleoprotoins;  /.  e.,  phospiiorus  and  iron.     If  rapidly 

'Lippert,  Frankf.  Zeit.  Path.,  1913  (14),  477. 

^Risel,  Verh.  Deut.  Path.  Gesell,  1909  (13),  322. 

'■An  "oil  cyst"  behind  the  ear  has  been  fully  analyzed  by  Kreis  (Schweiz. 
Apoth.  Ztg.,  1918  (56),  81)  and  found  to  contain,  in  addition  to  much  neutral 
fat  and  cholesterol,  consideral)le  amounts  of  high  unsaturated  hydrocarbons. 

*  Arch.  klin.  Chir.,  1880  (25),  49. 

'  Wien.  klin.  Woch.,  1890  (3),  551;  see  also  Zdarek,  Zeit.  phvsiol.  Chem., 
1908  (57)   461. 

«  Virchow's  Arch.,  1899  (156),  151. 


MALIGNANT  TUMORS  523 

growinp;,  thoy  contain  much  potassium;  if  un(lcrp;oinp;  much  retrogres- 
sion, httlo  potassium  and  a  hirger  amount  of  calcium  (Beebe,  Clowes 
and  Frisbie).  On  account  of  the  extensive  disintegration,  the  products 
of  autolj^sis  are  usually  mu(^h  more  abundant  than  in  b(mign  tumors. 
The  composition  varies  greatly  with  the  origin,  although  to  a  less 
extent  than  with  the  benign  tumors.  In  Fraenkel's  laboratory^  it  was 
found  that  cancers  are  often  defective  in  tryptophane,  and  from  a 
squamous  cell  carcinoma  of  the  skin  little  or  none  of  this  amino-acid 
could  be  obtained,  although  normal  squamous  epithelium  is  rich  in 
trypto[:)hane.  Fasal,^'^  however,  found  usually  a  high  tryptophane 
jBgure  in  cutaneous  epithelioma,  but  very  irregular  results  in  other 
tumors.  As  Bang  and  Beebe  have  shown,  the  tumors  arising  from 
lymphatic  tissues  show  the  chemical  characteristics  of  these  structures, 
and  contain  histon  nucleinate.  Tumors  from  squamous  epithelium 
develop  keratin  in  direct  proportion  to  the  amount  of  maturity  the 
cells  reach.  Even  the  most  complex  and  specific  products  of  metabolic 
activity  may  be  developed  by  malignant  tumors  (e.  g.,  thyroiodin, 
epinephrine,  bile),  and  in  a  form  and  condition  capable  of  performing 
function.  As  Buxton  and  others  have  shown,  malignant  tumors  pro- 
duce a  great  variety  of  intracellular  enzymes.  The  idea  that  glycogen 
is  present  in  tumors  in  proportion  to  their  malignancy  has  been  dis- 
proved by  Lubarsch,  Gierke,  and  others;  among  the  malignant  tumors 
glycogen  is  found  particularly  in  chorioepitheliomas,  hypernephromas, 
and  squamous  cell  carcinomas.  Of  particular  importance  is  the  ob- 
servation of  Beebe,  that  the  composition  of  metastatic  growths  is 
modified  by  the  organ  in  which  they  are  growing,  so  that  they  tend  to 
resemble  the  organ  serving  as  their  host;  which,  however,  does  not  hold 
for  certain  of  their  enzymes  (Wells  and  Long).  In  a  case  of  primary 
carcinoma  of  the  liver,  Wolter'^  found  the  tumor  tissue  richer  in 
nuclein  phosphorus  and  poorer  in  phosphatids  than  the  adjacent  liver 
tissue;  cholesterol  was  0.25  per  cent,  of  the  fresh  weight,  fatty  acids 
1.67  per  cent,  and  water  82.33  per  cent.,  the  water  of  the  normal 
tissue  being  79.34  per  cent. 

As  to  the  special  varieties  of  malignant  growths,  there  is  little  as 
yet  determined  concerning  their  chemistry  beyond  what  has  been 
stated  previously.  The  variations  in  compositon  of  tumors  are 
largely  the  direct  result  either  of  their  resemblance  to  some  normal 
tissue  or  of  degenerative  changes  that  they  have  undergone. 

"  Colloid"  carcinoma  may  be  mentioned  specially,  in  view  of  the 

confusion  caused  by  the  lax  use  of  the  term  "colloid"  {q.  v.).     The 

fluid  contents  of  colloid  cancers  of  the  gastro-intestinal  tract  are 

usually   chiefly  epithelial   mucus,    containing   mucin   mixed   with   a 

greater  or  less  quantity  of  proteins  from  degenerated  cells  and  serous 

effusion.     This  mucin  is  acid  in  reaction,  is  precipitated  by  acetic  acid, 

9  Wien.  klin.  Woch.,  1912  (25),  1041. 
'»  Biochem.  Zeit.,  1913  (55),  88. 
11  Biochem.  Zeit.,  1913  (55),  2G0.  ■    , 


524 


THE  CHEMISTRY  OF  TUMORS 


and  has  an  affinity  for  basic  dyes.'^  The  colloid  cancers  of  the  mam- 
mary gland,  in  which  the  "colloid  degeneration"  involves  the  stroma, 
probably  contain  a  connective-tissue  mucin  analogous  to  that  of  the 
umbilical  cord,  as  also  do  the  myxosarcomas,  if  we  may  judge  by 
their  origin  and  staining  reactions,  but  no  exact  chemical  study  of 
these  substances  can  be  found.  Colloid  cancers  of  the  ovary,  arising 
usually  from  the  same  structures  as  the  ovarian  cysts,  contain  pseudo- 
mucin  or  alhed  bodies  (see  ''Ovarian  Cysts").  Colloid  tumors  of 
thyroid  tissue  often  contain  the  typical  colloid  of  normal  thyroid  tissue, 
even  when  metastatic  in  other  organs;  in  the  tumor-colloid  may  be  a 
relatively  normal  proportion  of  iodin  (Gierke  ^■^). 

Hypernephromas  possess  several  interesting  chemical  features. 
For  example  Gatti^'*  brought  forward  the  fact  that  such  a  tumor 
analyzed  by  him  contained  3.4735  per  cent,  of  lecithin,  which  agreed 
very  well  with  the  amount  of  lecithin  in  normal  adrenals.  Beebe^^ 
found  in  the  watery  extract  of  a  hypernephroma  the  following  sub- 
stances: tryptophane,  proteoses,  glycogen,  leucine,  and  tyrosine,  in- 
dicating the  occurrence  of  autolysis.  About  29  per  cent,  of  fat  was 
present,  which  was  all  extractable  without  pepsin  digestion,  and  the 
fat  contained  about  18  per  cent,  of  its  weight  as  cholesterol.  Lecithin 
was  also  present,  but  not  quantitatively  determined.  A  study  of  the 
fats  and  lipoids  of  hypernephromas  and  other  tumors  gave  the  results 
shown  in  the  following  table i^*^ 


"3 
a 

0) 

u 

c3 

a 

u 
o 

Hypernephromas 

°S3 
ll 
fa 

Carcinoma  of 
breast 

Sarcoma,  second- 
ary, inlUvei 

1 

2 

3 

4 

36.3 
7.6 

11.9 

20.6 
11.8 

18.4 

33.0 

28.0 
4.6 

6.4 

16,9 
6.0 

8.3 

22.7 

33.0 
6.7 

10.0 

20.4 
9.0 

13.4 

27.5 

38.4 
8.7 

14.0 

22.9 
8.3 

13.4 

21.4 

85.0 
0.5 

3.3 

0.7 
2.0 

13.3 

2.4 

8.6 
2.2 

2.4 

26.1 
1.7 

1.9 

20.0 

21.4      14.5 

Cholesterol,  per  cent,  total 'dry  weight 
Cholesterol,    per    cent,    dry,    fat-free 

0.9  1.6 
1.2        1.9 

Cholesterol,     per    cent,     ether-soluble 
substance 

4.3      11.0 

Lecithin,  per  cent,  total  dry  weight .  .  . 
Lecithin,   per  cent,  dry,  fat-free  sub- 

0.7  j  6.2 
0.9        7.3 

Lecithin,  per  cent,   ether-soluble  sub- 
stance   

3.0      39.8 

Hypernephroma  No.  1. — Typical  specimen,  with  the  usual  amount  erf  hemorrhage  and  necrosis; 
cells  much  vacuolated. 

Hypernephroma  No.  2. — Similar^tolNo.  1. 

Hypernephroma  No.  3. — Primary  growth  resembled  more  a  papilloma  than  an  ordinary  hyper- 
nephroma in  most  places;  no  vacuolization  of  cells,  little  necrosis,  and  no  hemorrhage. 

Hypernephroma  No.  4, — -Tumor  resembling  a  lipoma,  witli  a  stroma  in  places  resembling 
a  fibrosarcoma  in  structure.  In  only  a  few  areasj^were  cells  present  resembling  adrenal  tissue 
most  of  the  tissue  resembling  fatty  areolar  tissue. 

i-Thc  fluid  of  a  colloid  cancer  of  the  peritoiioviin  examined  by  llnwk  contained 
a  protein  resembling  serosa  mucin,  containing  11.5  \ivv  cent,  of  X  and  O.S  per 
cent,  of  8.     (McCrae  and  Coplin,  Amcr.  Jour.  Med.  Sci.,  1910  (151),  -175.) 

13  Hofmeistcr's  Beitr.,  1902  (3),  28(5. 

'*  Virchow's  Arch.,  1897  (150),  417. 

"Amer.  Jour.  Physiol.,  1904  (11),  139. 

"Wells,  Jour.  Med.  Res.,  1908  (12),  4G1;  see  also  Steinke,  Frankfurt.  Zeit. 
Path.,  1910  (5),  1G7. 


I 


MYELOPATHIC  I'liDTI-:! WRI A  525 

It  will  be  at  once  observed  that  the  two  typical  hypcrnoi)hromas,  Xos.  1  and  2, 
show  a  marked  resemblance  to  the  normal  adrenal  in  the  proportion  of  fat  and 
lipoids.  (The  lower  figure  for  lecithin  in  No.  1  probably  is  due  to  the  fact  that 
this  specimen  had  been  preserved  lon<;er  than  the  others.)  This  was  what  was 
to  be  expected  from  the  microscojjic  resemblance  of  these  tumors  to  adrenal 
tissue,  and  corroborates  the  results  of  Gatti's  and  Beebe's  observations  on  iso- 
lated cases.  More  surjirisinf";  is  the  fact  that  equally  comparable  results  were 
obtained  in  the  hypernephroma  (No.  3),  which  contained  only  cells  free  from 
vacuolization  and  not  at  all  resembling  adrenal  cells.  From  this  it  may  be 
concluded  that  in  these  tumors  of  adrenal  origin  the  amount  of  fats  and  lipoids 
present  cannot  be  estimated  from  the  degree  of  cytoplasmic  vacuolization  of  the 
cells,  or  the  extent  of  necrosis;  the  fatty  materials  are  an  integral  part  of  the  cells, 
present  in  them  as  an  essential  constituent  and  not  as  the  result  of  degeneration. 

The  results  of  analysis  of  two  carcinomas  and  a  sarcoma  indicate  that  the 
hypernephromas  are  peculiar  in  their  close  resemblance  to  adrenal  tissue  in  respect 
to  fat  and  to  lipoid  content.  The  amount  of  all  these  constituents  in  these  three 
tumors  is  far  below  that  found  in  the  hypernephromas,  although  in  the  carcinoma 
of  the  breast  the  amount  of  simple  fats  is  relatively  large,  as  might  be  expected 
in  view  of  the  function  of  the  cells  from  which  it  arose.  It  is  interesting  to  note 
that  a  carcinoma  of  the  gall-bladder  shows  a  rather  high  proportion  of  its  fatty 
material  as  cholesterol,  for  this  observation  may  bear  a  relation  to  the  well-known 
tendency  of  the  ei)ithclium  of  the  gall-bladder  to  form  cholesterol.  The  large 
proportion  of  lecithin  in  the  sarcoma  of  the  liver  may  possibly  be  due  to  the  in- 
fluence of  the  soil  upon  which  the  tumor  was  growing,  but  we  need  more  informa- 
tion concerning  the  lipoid  content  of  other  malignant  tumors  arising  in  different 
sites. 

Renal  hypernephromas  reproducing  the  adrenal  cortex  in  struc- 
ture do  not  contain  epinephrine,^^  but  tumors  of  the  adrenal  arising 
in  the  medulla  may  do  so.^*  Microscopically,  hypernephromas  con- 
tain much  glycogen.  The  special  tests  for  hypernephroma  tissue 
recommended  by  Croftan  seem  not  to  be  specific. ^^ 

Melanotic  tumors  produce  melanin,  which  seems  not  to  differ 
at  all  from  the  melanin  found  in  normal  pigmented  structures.  Hel- 
man-°  found  as  high  as  7.3  per  cent,  by  weight  of  melanin  in  melano- 
sarcomas.  (See  also  Melanin,  p.  474,  and  Enzymes  in  Tumors,  p.  505. 
Concerning  Chloromas-^  see  p.  480.) 

Multiple  Myelomas  and  Myelopathic  "Albumosuria" 

Multiple  mijelom.as  are  of  particular  chemical  interest,  because  of 
the  appearance  in  the  urine  in  such  cases  of  the  peculiar  protein  first 
described  as  an  albumose  by  Bence-Jones,^^  and  now,  because  of  lack 
of  grounds  for  its  definite  classification,  generally  known  as  the 
'^ Bence-Jones  body"  or  " Bence-Jones  protein."  Because  of  the  ex- 
tensive bone  destruction  there  is  also  an  excessive  excretion  of  cal- 
cium,-^ and  sometimes  metastatic  calcification  may  occur.  ^^     This 

1^  Greer  and  Wells,  Arch.  Int.  Med.,  1909  (4),  291;  Brooks,  Jour.  Exp.  Med., 
1911  (14),  550;  Ciaccio,  Deut.  Zeit.  f.  Chir.,  1910  (104),  277. 

18  Wegehn,  Verb.  Deut.  Path.  Ges.,  1911  (15),  255. 

19  Koerber,  Yirch.  Arch.,  1908  (192),  356. 

20  Arch,  internat.  Pharmacodyn.,  1903  (12),  271. 

2'  Metabolism  in  chloroma  does  not  differ  from  leukemia  (Sakaguchi,  INlitt. 
Med.  Fak.,  Tokio,  1914  (13),  198). 

22  For  Hterature,  see  Rosenbloom,  Biochem.  Bulletin,  1911  (1),  161;  Vance, 
Amer.  Jour.  Med.  Sci.,  1916  (152),  693. 

23  Blatherwick,  Amer.  Jour.  Med.  Sci.,  1916  (151),  432. 

24  Tschistowitsch  and  Kolessnikoff,  Virchow's  Archiv..  1909  (197),  112. 


526  THE  CHEMISTRY  OF  TUMORS 

variety  of  tumor  differs  from  the  standard  types  of  malignant  tumors 
in  thac  it  involves  the  marrow  of  many  bones  simultaneously,  in  a  very 
diffuse  manner,  without  usually  giving  evidence  of  a  true  metastasis. 
In  many  respects  it  resembles  the  leukemias,  pseudoleukemia,  and 
chloroma,  and  it  is  extremely  uncertain  as  to  where  in  the  classifica- 
tion of  tumors  and  of  the  diseases  of  the  blood-forming  organs  this 
disease  should  be  placed.  Histologically,  the  tumors  show  evidence  of 
being  derived  from  the  specific  cells  of  the  marrow,  either  from  the 
plasma  cells  (Wright)  or  from  the  neutrophile  myelocytes-^  or  their 
predecessors  (Muir).  Cases  of  myeloma  without  the  proteinuria 
have  been  described,  and  also  a  few  instances  of  the  presence  of 
apparently  typical  Bence-.Iones  protein  in  the  urine  without  myelomas, 
but  with  bone  carcinomas,  leukemia  or  chloroma.-'' 

Properties  of  the  "Bence=Jones  Protein." — Not  to  go  into 
details,  which  are  given  in  the  hterature  cited,  the  important  facts  con- 
cerning the  "  Bence-Jones  protein,"  and  its  appearance  in  the  urine 
{"myelopathic  albumosuria,"  Bradshaw),  are  as  follows: 

It  is  a  protein,  the  exact  nature  of  which  has  not  been  determined; 
at  first  considered  an  albumose  because  of  its  peculiar  reactions  to 
heat,  its  nature  has  since  been  contested,  but  the  weight  of  evidence 
seems  to  be  in  favor  of  the  contention  of  Simon  that  it  is  most  closely 
related  to  the  water-soluble  globuHn  of  the  blood.  In  certain  cases 
it  partly  precipitates  spontaneously  from  the  urine,"  and  it  may 
crystallize  in  the  renal  tubules. ^^  Its  most  characteristic  properties 
are  the  following: 

The  coagulation  temperature  is  low,  varying  from  49°-60°  in  various  cases, 
and  being  considerably  modified  by  the  amount  of  salts  and  urea  present  in  the 
solution.  Probably  the  protein  forms  a  molecular  compound  with  the  salts 
which  is  more  stable  at  100°  than  at  lower  temperatures  (Hopkins  and  Savory). 

In  many  cases  the  coagulum  is  redissolved  on  heating,  and  reappears  on  cool- 
ing, but  this  characteristic  feature  is  not  always  present,  and  often  disappears  in 
cases  where  at  first  it  is  present. 

A  precipitate  is  formed  by  strong  (25  per  cent.)  nitric  acid,  which  disappears 
on  heating  and  reappears  on  cooling.  Strong  hydrochloric  acid  causes  a  dense 
precipitate,  which  is  quite  typical  (Bradshaw). 

No  precipitate  is  produced  by  acetic  acid,  even  in  excess,  and  the  addition  of 
acetic  acid  to  a  hot  coagulated  specimen  causes  prompt  solution  of  the  coagulum. 

UnUke  albumoses,  this  substance  does  not  dialyze;  the  salt-free  solution  left 
in  the  dialyzing  bag  does  not  precipitate. 

A  purplish-violet  color  is  usually  given  with  the  biuret  reaction,  but  it  may 
be  more  reddish  in  color,  especially  if  little  copper  is  present. 

"  Forman  and  Warren  (Jour.  Cancer  Res.,  1917  (2),  79)  found  the  cells  to  con- 
tain granules  giving  the  indol-phenol  blue  reaction  and  hence  belonging  to  the 
myeloid  group. 

^'^  Glynn  has  described  a  glycoprotein  resembling  Morner's  body,  in  the  urine 
during  myeloma  (Liverpool  Med.  Chir.  Jour.,  1914,  p.  82).  A  crystallizable  pro- 
tein, resembling  the  Bence-Jones  body,  has  been  found  in  the  urine  of  a  woman 
with  gastric  cancer  without  any  bone  involvement  (Schumni  and  Kimmcrle,  Zeit. 
physiol.  Chem.,  1914  (92),  1). 

"  Rosenbloom,  Arch.  Int.  Med.,  1912  (9),  255. 

"  Loehlein,  Cent.  allg.  Path.,  1913  (24),  953. 


BENCE-JONES  PROTEIN  527 

Sulphur  is  readily  split  off  by  alkalies,  reacting  with  lead  acetate  to  produce 
lead  sulphide  (Boston). 

After  standinp;  in  alcohol,  by  which  the  protein  is  precipitated,  it  loses  its  solu- 
bility (differing  in  this  respect  from  albumose). 

As  to  the  exact  nature  of  this  protein,  httle  can  be  said  at  the  pre- 
sent time.  Since  protoproteoses,  dcuteroprotcosos,  and  peptone  are 
split  off  on  digestion  with  pepsin,  the  molecule  is  evidently  larger  than 
that  of  any  of  the  albumoses.  The  well-purified  substance  is  free 
from  phosphorus,  and  hence  contains  no  nucleins;  but  it  contains  con- 
siderable sulphur  (between  1  and  2  per  cent.),  which  is  readily  split 
off.  Like  casein,  it  contains  no  hetero-group  (lack  of  heteroproteoses 
on  digestion),  but  differs  in  containing  a  carbohydrate  group  (in  small 
amount)  and  in  the  absence  of  phosphorus.  On  hydrolysis  Magnus- 
Levj^'-^  obtained  glutaminic  acid,  tyrosine,  and  leucine,  but  no  glycine. 
He  found  the  nitrogen  distributed  as  follows:  amid-nitrogen,  9.9 
per  cent.;  humin-nitrogen,  9.8  per  cent.;  diamino-nitrogen,  6.4  per 
cent. — which  last  was  composed  of  :  histidine,  0.9  per  cent.;  arginine, 
2.4  per  cent.;  lysine  3.0  per  cent.  The  extensive  analytic  studies  of 
Hopkins  and  Savory^°  show  that  the  amino-acid  grouping  is  that  of  a 
typical  protein,  with  a  liigh  proportion  of  aromatic  radicals,  similar 
proteins  not  being  found  in  the  tumors  or  muscles  of  a  typical  case. 
In  fact,  the  amino-acid  content,  as  given  below,  indicates  that  Bence- 
Jones  protein  is  as  distinct  from  other  proteins  in  chemical  composition 
as  in  its  physico-chemical  properties.  The  amino-acids,  in  round 
numbers,  were  isolated  in  the  following  percentage  proportions  of  the 
entire  protein:  Valine-leucine  fraction,  14;  glutamic  acid,  8;  aspartic 
acid,  2;  proline,  2.7;  phenylalanine,  4.8;  t3T0sine,  4.2;  tryptophane, 
0.8;  cystine,  0.6;  arginine,  6;  histidine,  0.8;  lysine,  3.7;  sulphur,  1.2. 
An  important  point  in  this  work  is  the  agreement  in  composition  of 
the  proteins  from  two  different  cases,  being  identical  within  the  hmits 
of  the  analytic  methods,  showing  that  the  protein  is  of  constant  and 
characteristic  properties. 

Occurrence  of  "Myelopathic  Albumosuria." — Not  all  eases  of 
multiple  myeloma  show  the  presence  of  Bence-Jones  protein  in  the 
urine,  however,  and  it  is  present  occasionally  in  other  conditions. 
Multiple  bone  involvement  by  other  tumors  does  not  often  cause 
"albumosuria.""  There  is  no  evidence  that  it  occurs  in  the  normal 
body,  even  in  the  bone-marrow,  or  that  it  is  produced  as  a  step  in  the 
splitting  of  any  form  of  proteins.  A  few  cases  of  supposed  osteomala- 
cia have  been  reported,  with  the  Bence-Jones  bodj^in  the  urine,  but  on 
more  careful  investigation  these  seem  to  have  been  unrecognized  mye- 

'9  Zeit.  physiol.  Chem.,  1900  (30),  200. 

'«  Jour,  of  Physiol.,  1911  (42),  189. 

''  A  case  of  this  kind  has,  however,  been  described  by  Oerum  (Ugeskrift  f. 
Lager.,  1904,  No.  24),  in  which  the  bone  tumors  were  multiple  metastases  of  a 
gastric  carcinoma.  See  also  Boggs  and  Guthrie,  Amer.  Jour.  Med.  Sci.,  1912 
(144),  803. 


528  THE  CHEMISTRY  OF  TUMORS 

lomas  (e.  g.,  the  cases  of  Bence-Jones  and  of  Jochmann  and  Schumm). 
Similarly  the  case  reported  by  Askanazy  as  leukemia  with  Bence- 
Jones  protein  in  the  urine,  on  reexamination  was  found  to  be  multiple 
myeloma.  However,  at  least  eight  eases  of  true  chronic  leukemia  with 
Bence-Jones  proteinuria  have  been  reported.^-  Coriat^^  describes 
a  substance  found  in  a  pleuritic  fluid  which  gave  the  reactions  of  the 
Bence-Jones  body,  and  he  believes  that  it  may  have  been  formed  from 
serum  globulin  through  the  digestive  action  of  the  leucocytes  or  bac- 
teria, Zuelzer  reports  finding  the  same  body  in  the  urine  of  a  dog 
poisoned  with  pyridin.^'*  It  is  a  striking  fact  that  the  kidneys  elim- 
inate such  great  quantities  of  this  protein  without  being  permeable 
to  the  very  similar  normal  blood  proteins,  and  usually  without  show- 
ing evidence  of  structural  changes.  Also  that  when  injected  into 
animals  it  does  not  escape  freely  in  the  urine  as  it  does  in  man.  It 
may  be  found  in  the  blood  and  exudates  of  patients  with  myeloma, ^^  as 
much  as  7.8  per  cent,  having  been  found  in  the  blood  by  Jacobson.^^ 
Miller  and  Baetjer"  report  finding  a  protein  corresponding  closely  to 
Bence-Jones  protein  in  the  urine  of  three  apparently  normal  persons 
and  in  two  cases  of  hypertensive  nephritis  without  evidence  of  bone 
disease,  thus  opening  the  question  as  to  whether,  after  all,  this  protein 
is  invariably  associated  with  bone  disease.  Simon^^  has  observed  that 
the  protein  may  be  accompanied  by  dialyzable  substances  giving  the 
ninhydrin  reaction,  probably  amino-acids  or  peptids. 

Origin  of  the  Protein. — As  to  the  place  of  formation  of  this  pe- 
culiar protein,  there  is  much  diversity  of  opinion.  Magnus-Levy 
advanced  against  the  idea  that  it  is  formed  by  the  tumor  cells,  the 
following  arguments:  In  the  urine  of  myeloma  patients  are  excreted 
great  quantities  of  the  protein,— as  much  as  30  to  70  grams  per  day, 
— whereas  the  total  amount  of  protein  in  all  the  tumor  tissue  in  the 
body  seldom  exceeds,  or,  indeed,  equals  this  quantity.  It  seems  im- 
probable that  so  little  tumor  tissue  can  form  so  much  urinarj^  protein, 
and  Magnus-Levy  suggests  that  it  must  come  from  the  food  proteins 
as  a  result  of  altered  protein  metabolism.  Against  this  view,  however, 
are  the  following  facts:  (1)  The  Bence-Jones  body  has  been  found 
(but  not  constantly)  in  the  myeloma  tissue,  but  not  in  other  organs 
or  tissues;  (2)  the  quantity  in  the  urine  is  not  dependent  upon  diet; 
(3)  it  is  associated  almost  exclusively  with  this  form  of  tumor.  Simon 
considers  it  probable  that  the  protein  is  formed  from  serum-globulin, 
perhaps  by  an  enzymatic  action  of  the  tumor  cells,  and  once  formed, 
it  is  rapidly  eliminated  by  the  kidneys,  as  are  all  foreign  proteins. 

32  Boggs  and  Guthrie,  Bull.  .Johns  Hopkins  Hosp.,  1913  (24),  368. 
"  Amer.  Jour.  Med.  Sci.,  1903  (12G),  631. 

'*  Wolgemuth  (Arb.  a.  d.   Path.  Inst,  zu    Berlin,    Festschrift,   190(5,   p.  027) 
states  that  normal  human  bone  marrow  may  contain  true  albumosos. 
"Taylor    el    al,    Jour.     Biol.     Chem.,     1917    (29),    425. 
"^  Jour.  Urol.,  1917  (1),  167. 
"  Jour.  AiiuT.  Med.  Assoc,  1918  (70),  137. 
".Jour.  Anicr.  Med.  Assoc,  1918  (70),  224. 


BENCE-JONES  PROTEIN  529 

Normal  bone  marrow  does  not  contain  this  protein  (Nerking^'*). 
Roscnbloom'"'  has  found  evidence  that  Bence-Joncs  protein  may  pos- 
sibly be  derived  from  the  osseo-albunioid  of  the  bones.  Weber  and 
Ledin<j;ham''^  have  suggested  that  it  comes  from  the  cytophismic  resi- 
due of  kar3'olyzed  plasma  cells.  The  observation  that  under  benzol 
treatment  the  amount  of  Bence-Jones  protein  in  the  urine  of  leukemic 
patients  is  reduced  (Boggs  and  Guthrie^^)  is  also  good  evidence  of 
its  myelogenous  nature.  The  fact  that  Abderhalden  and  Rostoski''^ 
found  that  the  serum  of  rabbits  immunized  with  Bence-Jones  protein 
gives  the  precipitin  reaction  with  human  serum,  is  evidence  that  the 
protein  is  a  human  tissue  protein  and  not  merely  an  absorbed  and 
excreted  food  protein.  This  has  been  corroborated  b}'  Hopkins  and 
Savory/^  who  also  found  that  the  amount  of  protein  in  the  urine, 
which  contained  about  one-third  the  total  nitrogen  excreted,  varied 
with  the  general  metabolism  and  was  not  controlled  by  the  diet. 
Massini^^  reports  securing  positive  complement  fixation  tests  with 
immune  sera,  differentiating  the  Bence-Jones  protein  from  normal 
serum  proteins;  positive  sensitization  tests  were  not  obtained  by  cu- 
taneous injections  of  the  protein  by  Boggs  and  Guthrie.  Injected 
into  the  blood  it  is  non-toxic  and  does  not  lower  coagulability  as  a  pro- 
teose would.  It  is  capable  of  acting  as  an  antigen  in  anaphylaxis 
reactions,  which  also  indicates  that  it  is  a  complete  protein  and  not  a 
cleavage  product. ''^  When  injected  into  dogs  it  is  partly  utilized, 
although  nephritic  animals  excrete  it  partly  hydrolyzed  into  proteose.  ^^ 

'^  Biochem.  Zeit.,  1908  (10),  167;  corroborated  by  Hopkins  and  Savory,  Jour. 
Physiol.,  1911  (42),  189. 

"  Arch.  Int.  Med.,  1912  (9),  236. 

^1  Foha  Hematol.,  1909  (8),  14. 

«  Zeit.  physiol.  Chem.,  1905  (46),  125. 

'^  Corroborated  also  bv  Boggs  and  Guthrie,  Amer.  Jour.  Med.  Sci.,  1912  (144), 
80;^;  Folin  and  Denis,  Jour.  Biol.  Chem.,  1914  (18),  277. 

*^  Deut.  Arch.  khn.  Med.,  1911  (104),  29. 

«  Taylor  and  Miller,  Jour.  Biol.  Chem.,  1916  (25),  281;  1917  (29),  425. 


CHAPTER  XX 

PATHOLOGICAL    CONDITIONS    DUE    TO,    OR     ASSOCIATED 

WITH,   ABNORMALITIES   IN    METABOLISM,   INCLUDING 

AUTOINTOXICATION 

During  the  course  of  metabolism  innumerable  organic  compounds 
are  formed,  some  of  which  ai-e  of  a  more  or  less  poisonous  nature. 
As  long  as  the  body  is  in  a  normal  condition,  these  injurious  substances 
are  kept  from  accumulating  in  sufficient  quantities  to  do  harm;  this 
is  accomplished  in  one  of  the  following  ways:  (1)  elimination  from  the 
body  in  the  urine,  feces,  etc.;  (2)  combination  with  other  substances 
into  harmless,  or  relatively  harmless,  compounds;  (3)  chemical  al- 
teration into  compounds  that  are  non-toxic  or  relatively  innocuous. 
Therefore  a  harmful  accumulation  of  metabolic  products  may  be  the 
result  of  any  one  of  the  following  conditions: 

(1)  Failure  of  elimination  because  of  abnormal  conditions  in  the 
eliminating  organs;  e.  g.,  uremia. 

(2)  Failure  of  neutralization  by  chemical  combination,  presumably 
due  to  abnormalities  in  the  organs  or  tissues  through  whose  activities 
the  neutralization  is  normally  accomplished;  e.  g.,  diseases  of  the  liver. 

(3)  Failure  in  the  chemical  transformation  of  the  metabolic  prod- 
ucts; this  may  result  either  from  abnormalities  in  the  functionating 
tissues,  or  through  a  checking  of  the  normal  steps  of  metabolism  by 
the  failure  of  elimination  of  the  end-products. 

(4)  Excessive  formation  of  certain  normal  products  of  metabolism ; 
e.  g.,  hyperactivity  of  the  thyroid. 

(5)  Production  of  abnormal  toxic  chemical  substances;  e.  g.,  the 
intoxication  following  superficial  burns. 

Numerous  classifications  of  autointoxication  have  been  proposed 
by  various  authors,  some  excluding  from  the  causes  of  autointoxication 
all  but  the  products  of  metabohsm  within  the  blood  and  tissues  of 
the  body,  as  has  been  done  in  the  preceding  consideration;  manj'  in- 
cluding intoxications  caused  by  the  jiroducts  of  gastro-intestinal  fer- 
mentation and  putrefaction;  and  still  others  (v.  Jaksch)  including 
even  the  intoxications  produced  by  bacterial  invasion  of  the  body.^ 
It  is  extremely  difficult  to  draw  the  line  as  to  just  what  should  be 

1  See  r6sum6  by  Weintmud,  Ergeb.  der  Patli.,  1897  (4),  1. 

530 


AUTOINTOXICATION  531 

included  under  tlie  term  autointoxication,  and  jiarticularly  difficult 
to  decide  the  proper  placing  of  the  intoxication  resultinp;  from  fecal 
retention  and  from  processes  of  decomposition  in  the  alimentary 
canal.  For  example,  the  poisoning  following  the  eating  of  partially 
decomposed  canned  food  could  not  be  looked  upon  as  an  autointoxi- 
cation, and  yet  there  is  no  fundamental  difference  whether  the  decom- 
position occurs,  as  in  this  case,  before  the  food  enters  the  body,  or 
whether  it  occurs  in  the  intestinal  tract  because  of  abnormal  bacterio- 
logical or  anatomical  conditions.  On  the  other  hand,  since  many 
of  the  obnoxious  products  of  meta])olism  arc  eliminated  through  the 
bowels,  failure  of  elimination  through  this  channel  ma}'  lead  to  a  true 
autointoxication  as  much  as  may  deficient  renal  elimination.  On  the 
whole,  it  seems  best  to  restrict  the  term  autointoxication,  as  far  as 
possible,  to  the  disturbances  produced  by  products  of  metabolism 
that  have  been  formed  within  the  tissues  of  the  body  (intermediary 
Dietabolism) ,  considering  as  a  distinct  but  related  subject  gasti-o-in- 
testinal  autointoxication. 

In  the  discussion  of  autointoxication  from  the  standpoint  of  chemi- 
cal pathology,  we  are  interested  particularly  in  the  chemical  nature 
of  the  substances  that  cause  the  intoxication,  and  in  the  chemical 
processes  by  which  their  action  is  kept  at  a  mininmm,  rather  than  in 
the  clinical  features  or  anatomical  results  that  may  be  produced. 
Unfortunately,  in  but  a  few  instances  have  the  exact  chemical  sub- 
stances causing  these  intoxications  been  accurately  determined,  prob- 
ably because  in  most  cases  not  one  but  a  number  of  poisonous 
substances  are  present;  and,  furthermore,  we  do  not  always  know  ex- 
actly when  a  certain  disease  is  to  be  ascribed  to  autointoxication,  nor 
can  we  always  determine  that  the  cause  of  a  certain  intoxication  lies 
in  an  abnormality  in  metabolism  and  not  in  an  infection  of  hidden 
nature.  It  is,  therefore,  quite  impossible,  with  the  uncertain  informa- 
tion available  at  this  time,  to  consider  autointoxication  in  a  systematic 
way,  and  we  must  limit  ourselves  to  a  consideration  of  certain  patho- 
logical conditions  in  which  there  appears  to  be  an  element  of  abnormal 
metabolism  with  resulting  intoxication.  In  some  cases  this  intoxica- 
tion is  a  prominent  feature  of  the  disorder,  in  others  it  is  subordinate 
to  other  manifestations  of  the  disease;  and,  finally,  we  may  have 
marked  alterations  in  metabohsm  without  evidences  of  disturbance  of 
health  (e.  g.,  cystinuria,  alkaptonuria). 

Of  the  autointoxications  due  to  the  retention  of  poisonous  products 
of  metabolism  that  should  be  excreted  from  the  body,  first  in  order 
of  importance  stand  uremia  and  cholemia  (the  latter  has  already  been 
considered  in  connection  with  the  discussion  of  Icterus,  Chap,  xviii). 
Of  apparently  less  significance  are  autointoxications  due  to  failure  of 
elimination  of  gaseous  metabolic  products  by  the  lungs,  and  failure 
of  the  excretory  function  of  the  skin. 


532  ABNORMALITIES  IN  METABOLISM 

UREMIA2 

The  cause  or  causes  of  the  severe,  often  fatal,  intoxication  that 
may  occur  when  the  outflow  of  urine  is  completely  checked,  or  when 
it  is  qualitatively  and  quantitatively  altered  for  long  periods  of  time, 
have  not  yet  been  definitely  determined.  As  the  kidney  seems  to  be 
the  chief  organ  for  the  removal  of  the  products  of  nitrogenous  metab- 
olism, it  is  naturally  assumed  that  uremia  is  the  result  of  a  retention 
of  these  products,  but  as  yet  it  has  not  been  ascertained  which  of  the 
many  products  is  responsible,  and,  indeed,  there  are  very  good  reasons 
for  questioning  if  the  substances  present  in  normal  urine  do  or  can 
cause  uremia  when  their  elimination  by  the  kidney  is  defective. 
There  is  no  question  but  that  the  urine  contains  toxic  substances. 
Among  them  are  the  salts  of  potassium,  which,  however,  cannot  alone 
explain  all  the  urinary  toxicity,  for  the  symptoms  produced  by  the 
injection  of  urine  are  different  from  those  produced  by  potassium  salts, 
and  it  has  been  found  that  the  inorganic  constituents  (ash)  of  urine  are 
less  poisonous  than  the  entire  urine.  Furthermore,  toxic  mixtures  of 
organic,  ash-free  substances  have  been  obtained  from  normal  urine. ^ 
Of  the  known  normal  constituents  of  the  urine  there  are  few,  however, 
that  are  toxic  to  any  considerable  degree,  and  these  occur  in  but  very 
small  quantities.  Urea  is  generally  considered  as  almost  absolutely 
non-toxic,  the  animal  body  withstanding  injection  of  large  quantities 
without  appreciable  injury.  Uric  acid,  the  purine  bases,  hippuric 
acid,  creatinine,  and  the  urinary  pigments  are  all  possessed  of  ver}^ 
slight  toxicity,  and  their  effects  do  not  explain  uremia.  Injections  of 
urine  into  animals  may  cause  more  or  less  disturbance,  but  it  is  differ- 
ent, on  the  whole,  from  the  manifestations  of  uremia.  (The  experi- 
ments of  Bouchard  and  his  school  present  such  serious  errors  of  tech- 
nique and  interpretation  that  they  are   now  largely  disregarded.) 

For  these  and  other  reasons,  it  has  been  generally  considered  that 
the  intoxication  of  uremia  is  not  due  solely  or  chiefly  to  the  substances 
that  are  normally  eliminated  in  the  urine,  but  rather  to  more  toxic 
antecedents  of  the  nitrogenous  constituents  of  the  urine.  Urea  repre- 
sents but  the  final  product  of  a  long  series  of  reactions  by  which  the 
huge  protein  molecule  is  broken  up  into  its  "building-stones,"  the 
various  amino-acids,  and  these  in  turn  are  decomposed  in  such  a  way 
that  their  NH2  groups  are  combined  with  carbonic  acid"*  and  eliminated 

.NHo 

as  the  diamido-compound  of  carbonic  acid,  namely  urea,  0  =  C\ 

We  know  that  the  liver  is  able  to  accomi)lish  the  conversion  of  amino- 
acids  to  urea,  for  it  has  been  experimentally  shown  that  if  leucine  and 

^General  r6sum6  with  earlier  literature  by:  Honigmann,  Ergeb.  der  Pathol., 
1894  (Bd.  1,  Abt.  2),  639;  1902  (8),  549;  Ascoli,  Vorlesungen  iibcr  Uriimie,  Jena, 
1903. 

'  See  Drcsbach,  Jour.  Exp.  Med.,  1900  (5),  315. 

*  Arginine  alone  of  all  the  amino-acids  splits  off  urea  diroctlj'  from  its  molecule. 


UREMIA  533 

glycine  are  passed  through  the  vessels  of  the  isolated  liver  they  di-siq)- 
pear  in  part,  while  an  increased  amount  of  urea  escapes  from  the  he- 
patic veins.  It  is  probable  tiiat  t  lie  liver  is  the  chief  site  of  urea  formation, 
but  it  is  also  probable  that  urea  can  b(^  formed  in  other  organs.  We 
do  not  know,  however,  the  intermediate  steps  by  which  the  amino- 
acids  of  the  protein  molecule  are  converted  into  urea.  It  has  been 
repeatedly  shown  that  urea  can  be  formed  from  ammonium  salts  of 
organic  acids  (including  anunonium  carbonate),  and  ammonia  is  a 
constant  protluct  of  autolysis,  being  characteristically  more  abundant 
as  a  product  of  autolytic  proteolysis  than  as  a  product  of  tryptic  pro- 
teolysis; therefore,  one  of  the  antecedents  of  urea  is  probably  ammo- 
nia, which  is  somewhat  toxic  and  especially  hemolytic.^  Another 
antecedent  of  urea  is  ammonium  carbamate,  which  stands  in  structure 
intermediate  between  urea  and  ammonium  carbonate,  as  shown  by  the 
following  graphic  formulse: 

/OH  /O-NH4  /NH2  /NH2 

O  =  C<  O  =  C<  O  =  C<  0  =  C< 

\0H  \O-NH4  \O-NH4  \NH2 

(carbonic  acid)  (ammonium  carbonate)         (ammonium  carbamate)  (urea) 

That  ammonium  carbamate  is  possibly  an  important  precursor  of 
urea  has  been  shown  particularly  through  the  results  of  studies  of 
dogs  with  Eck's  fistula,^  which  consists  of  a  fistula  between  the  portal 
vein  and  the  inferior  vena  cava,  the  blood  from  the  portal  system 
then  passing  directly  into  the  general  circulation  without  first  passing 
through  the  liver.  In  such  animals  the  urine  becomes  poor  in  urea 
and  relatively  rich  in  ammonium  carbamate.  At  the  same  time,  the 
dogs  show  severe  symptoms  of  intoxication  from  which  they  die,  and 
which  are  similar  to  the  symptoms  that  follow  intravenous  injection 
of  ammonium  carbamate.  Ammonium  carbamate,  being  a  substance 
of  considerable  toxicity^  when  free  in  the  blood,  it  has,  therefore, 
been  quite  widely  considered  that  it  may  be  an  important  factor  in 
the  production  of  uremic  symptoms.  On  the  other  hand,  it  seems 
most  probable  that  the  condition  of  uremia  does  not  depend  upon 
one  but  upon  many  various  and  varying  substances,  especially  as 
Hawk^  found  that  sodium  carbamate  did  not  produce  uremic  symp- 
toms in  his  Eck  fistula  dogs,  while  Liebig's  extract  did.^  Clinicallj^, 
the  symptoms  of  uremia  in  different  cases  are  widely  different;  thus 
if  uremia  is  due  to  complete  suppression  of  urine  through  mechanical 

^  Concerning  the  toxicity  of  ammonium  salts  see  Rachford  and  Crane,  Medical 
News,  1902  (81),  778. 

«  See  Hahn,  Massen,  Nencki,  and  Pawlow,  Arch.  f.  exp.  Path.  u.  Pharm.,  1893 
(32),  161. 

'  See  Bickel,  "Exp.  Untersuch.  liber  Cholaemie,"  Wiesbaden,  1900. 

8  Amer.  Jour.  Physiol.,  1908  (21),  260. 

^  Fischler  believes  the  intoxication  which  occurs  after  feeding  meat  to  Kck 
fistula  dogs  to  be  an  alkalosis,  probably  from  NH3  salts  (Deut.  Arch.  khn.  ?.Icd., 
1911  (104),  300). 


534  ABNORMALITIES  IN  METABOLISM 

obstruction,  the  symptoms  are  quite  different  from  those  observed 
in  the  uremia  following  a  chronic  nephritis;  drowsiness,  weakness  of 
heart  action,  and  syncope  being  the  chief  manifestations  of  obstructive 
uremia,  the  convulsions  and  other  manifestations  of  nervous  irritation 
characteristic  of  uremia  in  chronic  nephritis  being  absent.^'' 

Chemical  Changes  in  Uremia.— The  attempts  to  isolate  from  the 
blood  and  organs  of  uremic  patients  or  animals  toxic  substances  that 
explain  the  manifestations  of  uremia  have  thus  far  failed.  That 
there  is  an  actual  retention  of  organic  substances  in  the  blood  in 
uremia  is  shown  conclusively,  however,  by  the  studies  of  the  phj^sico- 
chemical  properties  of  the  blood.  It  has  been  repeatedly  found  that 
in  uremia  the  freezing-point  of  the  blood  is  reduced  markedly  below 
the  normal ;^^  instead  of  the  normal  depression  of  0.55°-0.57°  the 
freezing-point  is  usually  reduced  more  than  -0.60°,  and  sometimes  as 
much  as  -0.75°,  which  shows  that  the  number  of  molecules  in  the  blood 
is  increased.  ^^  At  the  same  time,  the  electrical  conductivity  may  not 
be  at  all  increased  (Bickel),^^  but  may  even  be  reduced;  and  as  the 
electrical  conductivity  of  the  blood  depends  upon  the  number  of 
dissociable  molecules,  chiefly  inorganic  salts,  these  are  evidently  not 
increased.^'*  Therefore,  the  increased  number  of  molecules  must 
represent  an  excess  of  organic  molecules  that  dissociate  but  little  if 
at  all,  and  hence  are  not  conductors  of  electricity.  Some  authors, 
indeed,  have  ascribed  uremia  to  the  increased  osmotic  pressure  of 
the  blood  from  the  retained  molecules,  but  tliis  is  improbable,  accord- 
ing to  Strauss,'^  who  found  that  a  marked  increase  in  molecular  con- 
centration may  occur  without  uremia,  and  that  we  may  have  a  severe 
uremia  without  increased  osmotic  pressure. 

Careful  metabolic  studies  have  shown  that  nephritics  (chronic  inter- 
stitial) are  not  able  to  convert  proteins  into  urea  as  rapidly  or  as  com- 
pletely as  normal  persons. ^^  Erben'^  has  studied  the  variations  in 
the  normal  components  of  the  blood  during  nephritis,  and  found 
the  albumin  generally  decreased  in  proportion  to  the  globulin,  espe- 
cially in  cases  of  parenchymatous  nephritis;  lecitliin  and  calcium  are 
also  decreased.  Rowe^^  found  the  serum  proteins  greatly  lowered  in 
chronic  nephritis  with  uremia,  an  increased  proportion  of  globulin 
being  present;  with  uremia  the  total  protein  content  is  normal  or 
slightly  higher,  with  usually  increased  globulin,  while  nephritis  wilh- 

1"  Chiari,  however,  observed  true  uremia,  both  clinical  and  anatomical,  in  a 
man  with  ureteral  obstruction  (Verh.  Deut.  Path.  Gesell.,  1012  (15),  207). 

11  See  Tieken,  Amer.  Med.,  1905  (10).  pp.  393,  567,  and  822;  ButtorfioUl  et  al . 
Amer.  Jour.  Med  Sci..  1916  (151).  63. 

*"  See  table  of  freezing  points  of  blood  and  elTusioiis  on  paj^c  355. 

"  Deut.  med.  Woch.,  1902  (28),  501. 

"See  Bienenstock  and  Csaki,  Biocheni.  Zeit.,  1917  (84),  210. 

"  Die  chronischen  Nieronent/iindungen,  etc.,  Berlin,  1902. 

loLevene  el  al.,  Jour.  Expcr.  Med.,  1909  (11).  825. 

"Zeit.  klin.  Med.,  1903  (50),  441;  1905  (57),  39. 

18  Arch.  Int.  Med.,  1917  (19),  354. 


UREMIA  535 

out  edema  or  uremia  produces  a  marked  increase  in  the  globulin. 
The  decrease  in  red  corpuscles  and  hemoglobin  in  nephritis  is  a  well- 
known  feature.  Bloor"'  found  in  the  blood  high  fat  content  in  both 
corpuscles  and  plasma,  high  lecithin  in  the  corpuscles,  and  normal 
cholesterol,  these  changes  probably  depending  on  the  lowered  alkali 
reserve. 

Measurements  of  the  partial  pressure  of  CO2  in  the  alveolar  air 
in  uremia  indicate  a  certain  degree  of  acidosis.^"  This  seems  to  occur 
to  a  sufficient  degree  to  be  responsible  for  definite  clinical  symptoms 
of  acidosis  only  in  advanced  nephritis,  but  earlier  in  nephritis  an  aci- 
dosis may  be  demonstrable  by  the  alkali  tolerance  test  when  it  is  not 
suffi-cient  to  affect  the  alveolar  air.^'  The  maximum  degrees  of  acidosis 
found  in  uremia  are  about  equal  to  those  of  diabetic  coma,  and  may  be 
an  important  feature  of  uremia,  although  usually  the  convulsive  fea- 
tures of  uremic  coma  are  quite  different  from  the  air  hunger  of  diabetic 
coma. 

The  development  of  this  terminal  acidity;  together  with  the  finding 
of  albumose  in  the  blood  of  a  nephritic  by  Schumm,--  suggests  the 
probability  of  active  autolytic  processes  occurring  in  uremia.  Neuberg 
and  Strauss-^  have  also  found  glycine  in  considerable  quantities 
(1.5  per  mille)  in  the  blood-serum  of  a  uremic  patient  and  in  the  blood 
of  nephrectomized  rabbits.  The  amount  of  colloidal  material  present 
in  the  urine  is  decreased  in  nephritis,  according  to  Pribram,-*  who 
suggests  that  retention  of  this  material,  which  is  rich  in  aromatic 
radicals,  may  be  of  importance  in  the  toxicity  of  uremia.  Rumpf 
found  that  the  organs  of  nephritics  contain  an  excess  of  potassium, 
and  Blumenfeldt"  attributes  this  to  a  defective  elimination  of  potas- 
sium salts  which  he  observed  in  nephritis.  Basal  metabolism  is  some- 
what lowered. ^^ 

Numerous  attempts  have  been  made  by  both  chemical  and  immu- 
nological methods  to  determine  whether  the  proteins  in  the  urine  in 
nephritis  come  from  the  food,  the  blood,  or  from  the  renal  cells  them- 
selves. In  alimentary  albuminuria  the  urinary  proteins  seem  not  to 
be  those  of  the  food,  but  human  proteins.^'  In  nephritis  differentia- 
tion between  serum  proteins  and  kidney  proteins  has  not  yet  been 
satisfactorily  accomplished.-^ 

The  development  of  improved  methods  of  analysis  of  small  quan- 

"Jour.  Biol.  Chem.,  1917  (31),  575. 

20  Straub  and  Schlayer,  Munch,  med.  Woch.,  1912  (59),  569;  Whitney,  Arch. 
Int.  Med.,  1917  (20),  931. 

21  Peabody,  Arch.  Int.  Med.,  1915  (16),  955. 
"  Hofmeister's  Beitr.,  1^03  (4),  453. 

"  Berl.  klin.  Wocli.,  HiO)  (43),  2c8- 

-*  Fortschr.  d.  Med.,  1911  (29),  951. 

"  Zeit.  exper.  Pathol.,  1913  (12^,  523. 

2«  Aub  and  DuBois,  Arch.  Int.  Med.,  1917  (19),  865. 

"  Wells,  Jour.  Anier.  Med.  Assoc,  1909  (53),  863. 

28  Cameron  and  Wells,  .\rch.  Int.  Med.,  1915  (15),  746. 


536  ABNORMALITIES  IN  METABOLISM 

titles  of  blood,  and  other  fluids,  especially  by  Folin  and  Denis,  Marshall, 
and  Van  Slyke,  has  enabled  us  to  obtain  exact  knowledge  of  many  of 
the  chemical  changes  of  nephritis  and  uremia.-^  It  has  been  found 
that  the  normal  blood  contains  from  25  to  40  mg.  of  nitrogen  in  non- 
coagulable  form  in  each  100  c.c,  there  being  usually  about  5  mg. 
increase  after  meals,  and  ordinarily  about  one-half  of  this  nitrogen  is 
in  the  form  of  urea.^°  In  all  conditions  that  impair  renal  function, 
whether  renal  changes  or  circulatory  deficiency,  there  is  a  rise  in  this 
noncoagulable  nitrogen,  and  when  there  is  excessive  tissue  destruction 
there  may  also  be  a  sHght  rise  independent  of  renal  injury.  As  a  gen- 
eral rule,  but  with  some  exceptions,  the  amount  increases  with  in- 
creased renal  impairment,  the  highest  figures  being  seen  in  uremia,  in 
which  figures  as  high  as  460  mg.  have  been  obtained.  In  130  nephritics, 
Foster  found  the  average  to  be  84  mg.  of  nitrogen. 

Analyses  of  the  blood  in  600  cases  of  nephritis  by  Get  tier  and  St. 
George^^  have  given  the  following  figures  in  mg.  per  100  c.c.  of  blood: 

Normal  Nephritis 

Nonprotein  nitrogen 25  to  40  40  to  460 

Urea  nitrogen 10  to  18  20  to  375 

Creatinin 0.1  to  0.8  2  to    42 

Uric  acid 0.5  to  3.0  3  to    17 

Sugar 60  to  110  75  to  160 

Alkali  reserve — per  cent 53  to  80  40  to    75 

From  their  observations  these  authors  conclude:  All  the  waste 
nitrogen  products,  nonprotein  nitrogen,  urea,  creatinin  and  uric  acid, 
are  present  in  increased  amounts  in  cases  of  true  nephritis,  and  gener- 
ally, but  not  invariably,  present  in  greater  concentration  in  the  blood 
of  those  cases  which  are  primarily  considered  as  chronic  interstitial 
nephritis  (retention  nephritis").  The  degree  of  retention  (when  taking 
into  account  the  functional  efficiency  of  the  cardiac  muscle)  is  a  direct 
criterion  of  the  severity  of  the  lesion.  The  sugar  content  in  the  blood 
is  similarly  increased  in  nephritis,  and  more  marked  in  the  patients 
suffering  with  the  chronic  parenchymatous  form  of  the  disease.  The 
alkali  reserve  is  a  valuable  index  of  the  degree  of  acidosis  present. 
There  is  no  definite  lesion  of  nephritis  referable  to  a  certain  clinical 
picture. 

There  is  no  constant  relationship  between  the  blood  pressure  and 
the  nitrogen  figure,  but  functional  tests  usually  show  a  correspondence 
between  the  excretorj^  power  of  the  Iddncy  and  the  retention  of  meta- 
boHtes  in  the  blood.  The  symptoms  of  asthenic  uremia  are  rarely 
well  defined  when  the  concentration  of  urea  in  the  blood  is  less  than 

2^  Good  reviews  and  bibliographies  arc  givtMi  bv  Tileston  and  Comfort,  Arch. 
Int.  Med.,  1914  (14),  620;  Schwartz   and    McCiill,  ibid.,   1916  (17),  42;  Woods. 
ibid.,  1915  (16),  577;  Karsner,  Jour.  Lab.  Clin.  Med.,  1916  (1),  910;  Feigl,  Biochem 
Zeit.,  1919  (94),  84. 

^"  Sec  Kast  and  Wardell,  Arch.  Int.  Med.,  1918  (22),  581. 

••".Jour.  Amer.  Med.  Asso.,  1918  (71),  2033. 


UREMIA  537 

100  mg.  per  100  cc,  and  they  are  rarely  absent  when  the  concen- 
tration exceeds  200  nig.''-  With  these  high  blood  nitrogen  figures 
there  is  also  an  increase  in  nonprotein  nitrogen  in  the;  tissues  CFoster)^^ 
and  metabolism  studies  show  nitrogen  retention  of  consi(lera})le  degree, 
sometimes  over  1  gram  retention  when  the  intake  is  but  10  grams  per 
day. 

Along  with  the  other  nitrogenous  constituents  the  uric  acid  is  in- 
creased from  a  normal  2  to  3  mg.  up  to  7  to  10  mg.,  and  even  higher. 
Creatinin  rises  from  1  to  2  mg.  up  to  5  to  20  mg.^*  On  the  other 
hand  the  amino-acid  nitrogen  may  be  normal  in  the  blood  even  with 
extremely  high  nonprotein  nitrogen  figures,'^  although  sometimes  it  is 
much  increased,  as  high  as  30  mg.  amino  acid  N  having  been  found 
b}'  Bock^^  in  uremia  (the  normal  figure  being  7  mg.).  FeigP^  reports 
finding  as  high  as  125  mg.  amino-N,  with  frequently  60  to  85  mg.  The 
retention  of  various  substances  varies  directly  with  the  solubility  and 
diffusibility  of  the  substances,  so  that  with  renal  disease  we  first  get 
retention  of  uric  acid,  then  urea,  and  last  of  creatinin  (Myers). '^ 
Ammonia  nitrogen  may  show  a  slight  increase,  rising  in  half  of  Fos- 
ter's cases  from  the  normal  0.5  mg.  to  from  0.7  mg.  to  2.2  mg.  per  100 
cc.  Indicanemia  may  also  be  present  but  it  is  not  a  toxic  factor 
(Dorner)."  The  blood  normally  contains  about  0.05  mg.  per  100 
cc;  in  uremia  it  may  rise  to  0.2  mg.,  and  as  much  as  2.2  mg.  has  been 
found  in  one  case.^^ 

The  Pathogenesis  of  Uremia. — The  fact  that  the  highest  figures 
for  non-protein  nitrogen  are  usually  found  in  uremia  might  be  accepted 
as  proving  that  uremia  is  caused  by  poisoning  with  these  metaboHtes, 
were  it  not  for  certain  contradictory  observations. 

(1)  Occasionally  quite  typical  attacks  of  uremia  are  observed  without  high 
nonprotein  nitrogen  figures  for  the  blood,  even  as  low  as  28  mg.  having  been  re- 
corded in  a  fatal  case.^* 

(2)  Extremely  high  nonprotein  nitrogen  content  may  be  observed  without 
uremia.  Thus,  Tileston  and  Comfort  found  169  and  150  mg.  in  two  cases  of  acute 
intestinal  obstruction  without  uremic  symptoms,  and  similar  results  have  been 
obtained  in  bichloride  of  mercury  poisoning,'"*  and  mechanical  anuria.  The  occur- 
rence of  albuminuric  retinitis  also  seems  to  bear  no  relation  to  the  nitrogen  retention 
(Woods). 

"  Hewlett,  Gilbert  and  Wickett,  Arch.  Int.  Med.,  1916  (18),  636. 

"  Arch.  Int.  Med.,  1919  (24),  242. 

»*  See  Myers  and  Fine,  Arch.  Int.  Med.  1915  (16),  536;  1916  (17),  570. 

»5  Foster,  Arch.  Int.  Med.,  1915  (15),  356. 

36  Jour.  Biol.  Chem.,  1917  (29),  191. 

"  Deut.  Arch.  klin.  Med.,  1914  (113),  342;  Rosenberg,  Arch.  exp.  Path.,  1916 
(79),  260;  Tscherkoff,  Deut.  med.  Woch.,  1914  (40),  1713;  Rev.  M6d.  Suisse  Ro- 
mande,  1918  (38),  15. 

2«  Hass,  Deut.  Arch.  klin.  Med.,  1916  (119),  177. 

3^  There  are  few  who  would  go  to  the  extreme  of  Strauss  (Berl.  klin.  Woch., 
1915  (52),  368)  and  limit  the  term  uremia  to  cases  showing  a  high  non-i)rotcin 
nitrogen  in  the  blood,  no  matter  what  the  sj-mptomatology  and  pathology  may 
be.  A  totally  different  viewpoint  is  expressed  by  Reiss,  Zeit.  kUn.  Med.,  1914 
(80),  97,  424,  452. 

^»  See  Foster,  Arch.  Int.  Med.,  1915  (15),  754. 


538  ABNORMALITIES  IN  METABOLISM 

(3)  None  of  the  known  nitrogenous  constituents  of  the  urine  can  be  held  re- 
sponsible for  all  the  manifestations  of  typical  uremic  poisoning.  The  highest 
purine,  uric  acid  and  creatinine  concentration  in  a  given  case  may  occur  entirely 
independent  of  uremic  conditions/^  the  amino-nitrogen  is  not  increased  in  uremia, 
urea  is  not  supposed  to  be  toxic  in  this  degree  arid  uremia  may  occur  without  high 
urea  concentration  or  be  absent  when  there  is  much  urea  in  the  blood.  To  be  sure, 
an  unknown  toxic  substance  may  be  responsible,  but  in  some  cases  of  uremia  the 
total  non-protein  nitrogen  can  be  accounted  for  b}^  the  known  nitrogenous  com- 
ponents found  in  the  blood  (Foster). 

We  may  consider  one  of  the  following  alternatives: 

(1)  The  nerve  cells  may  be  made  hypersensitive  to  some  one  of  the 
known  constituents  by  the  excessive  amounts  of  the  other  metabolites. 
This  is  a  purely  speculative  hypothesis,  without  anj'-  actual  evidence 
in  its  support. 

(2)  The  portion  of  unidentified  nitrogen  usually  present  in  the 
blood  may  contain  a  specific,  highly  efficient  poison. 

In  support  of  this  hypothesis  is  the  finding  in  a  series  of  cases  that  the  pro- 
portion of  noncoagulable  blood  nitrogen  that  could  not  be  accounted  for  by  the 
known  nitrogenous  metabolites  seemed  to  vary  directly  with  the  severity  of  the 
symptoms  (Woods). ^^ 

Hartman*'  has  suggested  that  the  substance  which  causes  the  characteristic 
odor  of  the  urine  may  be  responsible  for  at  least  some  of  the  intoxication  of  uremia. 
This  substance,  which  he  has  isolated  and  described  under  the  name  "urinod," 
he  believes  to  be  a  cyclic  ketone  with  the  empirical  formula  CeHsO;  it  is  highly 
toxic,  and  causes  mental  symptoms.  This  important  observation  awaits  confirma- 
tion. 

Foster^*  has  described  the  finding  of  a  toxic  base  in  the  blood  of  uremics, 
absent  from  the  blood  in  other  conditions,  which  causes  death  of  guinea  pigs  with 
symptoms  suggestive  of  the  eclamptic  type  of  uremia.  Further  development  of 
this  work  is  also  awaited. 

(3)  Uremia  may  not  depend  on  intoxication  of  the  nerve  cells,  but 
upon  the  mechanical  effects  of  edema  involving  these  cells. 

One  of  the  striking  features  of  autopsies  of  uremics  is  often  the  "wet  brain" 
and  the  excessive  amount  of  cerebrospinal  fluid  which,  during  life,  may  lie  found 
to  be  under  a  heightened  pressure.  We  know  that  not  only  general  but  localized 
edemas  occur  in  nephritis,  and  that  localized  edema  in  the  brain  may  be  associ- 
ated with  and  apparently  responsible  for  paralyses,  convulsions,  hyperirritability 
and  mania.  The  wet  brain  of  nephritis  is  similar  to  the  wet  brain  of  acute  alco- 
holism and  delirium  tremens.  Oftentimes  the  nervous  symptoms  of  uremia  are 
distinctly  focal,  and  a  complete  hemiplegia  from  hemorrhage  may  be  exactly 
simulated;  convulsive  seizures  identical  with  those  of  brain  tumor  may  be  seen. 
It  is  extremely  difficult  to  explain  these  localizations  by  the  action  of  a  soluble 
poison,  and  simple  if  we  assume  a  local  edema.  It  is,  of  course,  as  difficult  to 
explain  the  localization  of  the  edema,  but  we  know  that  in  nephritis  localized 
edemas  do  occur,  so  we  have  a  basis  for  the  assumption  of  localized  cerebral 
edemas.  A  general  acidosis  (q.  v.)  is  usual  in  nephritis  and  marked  in  uremia''^ 
but  we  have  no  means  of  knowing  whether  local  acidosis  occurs  in  the  nervous 
system  that  may  be  responsible  for  local  edemas  according  to  Fischer's  hypothe- 
sis.     Or,  osmotic  effects  may  be  responsible,  in  view  of  the  demonstrated  high 

*i  Myers  and  Fine,  Jour.  Biol.  Chem.,  1915  (20),  391. 
"  Arch.  Int.  Med.,  1915  (16),  577. 
"Ibid.,  1915  (16),  98. 

**  Trans.  Assoc.  Amer.  Phys.,  1915  (30),  305. 

«  Henderson,  Bull.  Johns  Hopkins  Hosp.,  1914  (25),  141;  Peabody.  Arch.  Int. 
Med.,   1915  (16),  955;  Sellards,  "Principles  of  Acidosis,"  Harvard  Press,   1917. 


UREMIA  539 

osmotic  pressure  of  the  blood  in  uremia,  and  the  fact  that  the  life  of  nephrecto- 
niizcd  rabbits  is  prolonged  by  giving  them  water.*"  In  any  evcnt^  the  existing 
evidence  on  the  pathogenesis  of  uremia  does  not  exjjlain  it  on  a  toxicologic  basis, 
and  hence  the  alternative  explanation  of  cerebral  edema  must  be  taken  into  con- 
sideration. Ervin"  proposes  the  hypothesis  that  convulsions  occur  when  the 
blood  pressure  becomes  lower  than  the  intracranial  pressure  so  that  the  blood  sup- 
ply of  the  brain  is  reduced;  the  convulsion  raises  the  blood  pressure.  He  comments 
on  the  ditHculty  in  explaining  the  transient  character  of  uremic  convulsions  if 
caused  by  concentration  of  chemical  poisons. 

On  the  other  hand  the  pathologist  recognizes  evidence  of  systemic 
intoxication  in  uremia.  The  uremic  pericarditis  and  endocarditis, 
which  have  often  failed  by  ordinary  methods  to  yield  bacteria,  are 
apparently  toxic  processes.  The  diphtheritic  colitis  indicates  vicari- 
ous excretion  of  poisonous  substances.  Structural  changes  are  found 
in  cells  that  suggest  poisoning;  chromatolysis  of  the  cortical  ganglion 
cells  has  been  repeatedly  observed  in  uremia,  and  in  nephrectomized 
rabbits  Lewis*''"  found  acute  parenchymatous  and  fatty  degeneration 
of  the  myocardium  and  endothehal  cells  of  the  liver.  The  localized 
edemas  of  nephritis  often  show  a  fluid  of  the  character  of  an  exudate 
rather  than  a  transudate. 

It  would  seem,  despite  the  prevailing  opinion  to  the  contrary,  that 
it  is  entirely  possible  that  the  manifestations  of  uremia  may  be  caused 
by  the  known  nitrogenous  substances  that  the  Iddneys  have  failed  to 
excrete,  and  that  the  only  difficult  thing  to  explain  is  the  failure  of 
investigators  to  consider  the  time  element  in  experimental  intoxica- 
tions. The  presence  of  200  mg.,  and  upwards,  of  nonprotein  nitrogen 
per  100  c.c.  of  blood,  which  is  often  found  in  uremia,  indicates  that 
the  blood  plasma  that  is  bathing  the  tissue  cells  contains  somewhere 
between  0.5  and  1.0%  of  soluble  organic  substances,  a  strength  of 
solution  that  certainly  does  not  require  any  very  high  degree  of 
toxicity  when  continuously  maintained  at  this  concentration,  as  it  is  in 
nephritis.  The  reported  experimentally  determined  toxicities  with 
these  substances  have  only  represented  transitory  conditions  which  are 
entirely  dissimilar  to  the  actual  conditions  in  the  body.  They  corre- 
spond to  the  cases  of  high  nonprotein  nitrogen  in  the  blood  in  in- 
testinal obstruction,  bichloride  poisoning,  etc.,  in  which  absence  of  the 
uremic  symptom  complex  has  been  noted  and  remarked  upon.  To 
study  the  relation  of  uremia  to  retained  metabohtes  we  need  observa- 
tions on  their  effects  when  maintained  in  the  organism  for  long  periods 
at  the  concentrations  occurring  in  uremics  and  this  can  be  done  readily 
by  such  methods  as  have  been  devised  by  Woodyatt.^^  A  start  in  this 
direction  is  furnished  by  Hewlett,  Gilbert  and  Wickett,^-  who  found 
that  when  large  doses  (100  to  125  gm.)  of  urea  were  given  to  normal 
men  there  occurred  symptoms  comparable  to  those  of  asthenic  uremia. 

«  Couvee,  Zeit.  klin.  Med.,  1901  (54),  311. 
"  Jour.  Amer.  Med.  Assoc,  1918  (70),  1208. 
4""  Jour.  Med.  Res.,  1907  (17),  291. 

"Jour.  Amer.  Med.  Assoc,  1915  (65);  2067;  Jour.  Biol.  Chem.,  1917  (29), 
355. 


540  ABNORMALITIES  IN  METABOLISM 

which  appeared  only  when  the  urea  concentration  of  the  blood  had 
reached  levels  of  160  to  245  mg.  of  urea  per  100  c.c.  i.  e.,  just  the  con- 
centrations that  are  usually  seen  in  well  developed  uremia.  If  in  these 
experiments  of  brief  duration  such  marked  symptoms  were  produced 
by  urea,  what  striking  effects  must  be  expected  when  these  same  urea 
concentrations  are  continued  in  the  blood  for  days  and  weeks  at  a 
time.^^  We  must  find  out  what  results  not  only  from  urea,  but  from 
creatinine  and  uric  acid  kept  in  the  blood  at  the  concentration  found 
in  uremia  for  long  periods,  as  well  as  any  other  substance  that  may  be 
increased  in  the  blood  in  uremia.  An  experiment  of  a  few  minutes' 
or  hours'  duration  cannot  be  expected  to  duplicate  or  elucidate  a  con- 
dition of  weeks  duration.  In  chronic  diseases  our  experimental  in- 
vestigations must  be  of  some  reasonably  comparable  duration,  and  this 
principle  of  investigation  is  now  made  possible  by  Woodyatt's  methods. 
And  finally,  in  view  of  the  extremely  varied  symptomatologj^  of  renal 
incompetence,  we  must  recognize  that  it  is  highly  probable  that  in 
different  cases  these  symptoms  vary  because  of  different  conditions. 
In  one  case,  urea  may  be  the  chief  factor,  in  another  the  action  of  urea 
may  be  complicated  by  the  effects  of  acidosis  or  high  blood  pressure 
per  se,  while  in  others  cerebral  edema  may  be  the  chief  influence. 
Some  continental  writers  hold  that  there  is  a  true  uremic  picture  due 
solely  to  cerebral  edema  from  salt  and  water  retention,  occurring 
especially  in  the  young,  to  be  distinguished  from  the  uremia  of  nitro- 
genous retention,  and  from  a  pseudouremia  resulting  from  the  circula- 
tory disturbances  of  arteriosclerosis. ^°  Rowland  and  Marriott  caU 
attention  to  the  reduced  calcium  in  the  blood  in  uremic  acidosis,  and 
as  nervous  irritability  is  increased  by  reduction  of  calcimn  this  may  also 
be  a  factor  in  the  nervous  manifestations  of  uremia.  All  possible 
shades  of  cooperating  influences  may  be  expected  to  occur  when  the 
kidneys  fail,  and  to  explain  the  confused,  variable,  changing  picture 
of  the  uremic  state. ^^ 

TOXEMIAS  OF  PREGNANCY" 

Under  this  heading  are  included  eclampsia,  as  characterized  by 
convulsions  and  certain  anatomical  changes,  together  with  those  in- 
stances of  intoxication  with  similar  anatomical  changes  and  no  con- 

*^  However,  in  Salachians  the  normal  urea  content  in  the  blood  is  over  two 

per  cent,  and  ammonium  salts  exceed  ^^  NHs,  (See  A.  B.  Macallum,  Amer.  Jour. 

Med.  Sci.,  1918  (156),  1),  but  it  may  well  be  that  in  such  species  the  tissues  are 
adapted  to  their  environment. 

60  See  Haim  and  TchertkofT,  Rev.  M6d.  Suisse  Romande,  1918  (38),  15. 

"^  The  influence  of  a  hypothetical  internal  secretion  of  the  kidney  (Brown- 
Sequard)),  or  of  the  products  of  nephrolysis  (.Ascoli),  as  a  cause  of  uremia,  may  now 
be  considered  as  of  historical  interest  only.  (See  Pearce,  Arch.  Int.  Med.,  1908 
(2),  77;  1910  (5),  133.)  The  same  is  true  of  the  attempt  to  explain  the  high 
blood  pressure  as  the  result  of  adrenal  hypertrophy.  (Pearce,  Jour.  Exp.  Med., 
190S  (10),  735;  1910  (12),  128.) 

"  Review  and  bibliography  by  Ewing,  Amer.  Jour.  Med.  Sci.,  1910  (139),  829. 


PUERPERAL  ECLAMPSIA  541 

vulsions,  and  the  related  pernicious  vomiting  of  pregnancy.  Acute 
yellow  atrophy  of  the  liver  belongs  in  tiie  same  categor}',  although 
often  occurring  independent  of  pregnancy. 

ECLAMPSIA" 

In  many  respects  eclampsia  resembles  uremia;  so  much  so,  indeed, 
that  Frerichs  and  others  have  referred  to  eclampsia  as  "puerperal 
uremia."  Considering  it  as  a  simple  uremia  occurring  in  pregnancy, 
uremia  and  eclampsia  have  in  common  the  constant  occurrence  of 
renal  disturbance  with  albuminuria  and  decreased  elimination  of  urea, 
and  also  violent  convulsions  and  profound  coma  terminating  in  death. 
On  the  other  hand,  eclampsia  differs  greatly  from  uremia  in  the  ana- 
tomical changes  observed  in  the  organs  of  the  body  other  than  the 
kidneys;  these  are  of  such  a  nature  that  in  some  cases  it  becomes  diffi- 
cult to  distinguish  eclampsia  from  acute  yellow  atrophy  of  the  liver, ^* 
while  in  other  cases  the  picture  resembles  that  of  a  profound  bacterial 
intoxication,  so  that  numerous  authors  have  urged  that  eclampsia  is 
the  result  of  a  bacterial  infection.  At  the  present  time  the  cause  of 
puerperal  eclampsia  is  quite  unknown,  but  there  is  a  decided  ten- 
dency to  assume  that  poisonous  substances  are  developed  in  the  pla- 
centa or  fetus,  or  are  formed  in  the  body  as  a  reaction  of  the  maternal 
organism  to  the  foreign  fetal  elements.  These  theories  will  be  dis- 
cussed after  considering  the  known  facts  concerning  the  chemical 
changes  of  the  disease  that  have  been  reported  by  various  observers. 

Chemical  Changes  in  Eclampsia. — Urinary  changes  are  practi- 
cally invariably  present,  and  usually  they  are  profound,  although 
there  are  no  known  characteristic  qualitative  or  quantitative  differ- 
ences from  the  urinary  changes  of  puerperal  albuminuria  without 
eclampsia.  Proteins  are  abundant,  including  a  large  proportion  of 
globulin,  decreasing  rapidly  after  delivery  as  a  rule.  The  urea  is 
usuall}'-  very  low,  but  generally  increases  with  great  rapidity  after 
delivery,  until  two  or  three  times  the  normal  amount  is  passed  per 
day;  as  urea  and  ammonia  do  not  seem  to  be  greatl}^  increased  in  the 
blood,  this  has  been  interpreted  as  indicating  that  during  eclampsia 
there  is  an  accumulation  of  the  precursors  of  urea  in  the  system 
(Sikes).  However,  the  involution  of  the  uterus  itself  results  in  an 
increased  nitrogen  excretion  which  probably  accounts  for  much  if 
not  all  of  these  findings  (Siemens). ^^  There  is  an  excessive  elimina- 
tion of  nitrogen  in  the  form  of  ammonia,  which  is  said  to  be  due  to 
the  formation  of  abnormal  quantities  of  sarcolactic  and  other  organic 
acids  in  the  body,  which  are  combined  with  ammonia  in  the  blood  and 

^'  Literature  is  given  by  Pikes  in  The  Practitioner,  1905  (74),  pp.  478  and 
642;  L.  Zuntz,  Handb.  d.  Biochem.,  1909,  III  (I),  366;  Seitz,  Arch.  f.  Gvn.,  1909 
(87),  79. 

^^  Concerning  the  liver  changes  see  Konstantinowitsch,  Ziegler's  Beitr.,  1907 
(40),  483. 

"  Bull.  Johns  Hopkins  Hosp.,  1914  (25),  195. 


542  ABNORMALITIES  IN  METABOLISM 

eliminated  in  the  urine.  ^®  This  fact  has  led  many  to  look  with  favor 
upon  the  idea  that  eclampsia  is  due  to  an  acid  intoxication.  Other 
nitrogenous  urinary  constituents  may  also  be  increased,  so  that  the 
relative  proportion  of  nitrogen  eliminated  as  urea  is  often  greatly 
reduced.  It  is  said  that  the  toxicity  of  the  urine,  which  is  high  in 
normal  pregnancy,  is  increased  if  the  kidneys  are  not  impaired,  but 
decreased  if  their  permeability  is  impaired  by  nephritis,  the  character 
of  the  toxicity  being  such  as  to  indicate  that  it  is  from  substances  de- 
rived by  disintegration  of  proteins  (Franz).  The  proportion  of  sul- 
phur eliminated  in  an  unoxidized  form,  as  compared  with  that 
eliminated  as  SO4,  is  much  greater  than  normal.  These  findings  all 
indicate  that  oxidation  within  the  body  is  impaired.  There  is  more  or 
less  retention  of  chlorides,  but  there  is  nothing  characteristic  in  this.^^ 
In  spite  of  the  hepatic  lesions  of  eclampsia  the  tolerance  for  levulose 
was  not  found  impaired  by  Alsberg.^* 

The  nonprotein  nitrogen  of  the  blood  is  but  little  increased  in 
eclampsia,  and  not  to  the  extent  usually  seen  in  uremia,  and  it  bears 
no  definite  relation  to  the  severity  of  the  symptoms  (Farr  and  Wil- 
liams).^^  They  found  from  25  to  72  mg.  per  100  c.c.  in  seven  cases. 
These  figures  can  be  reasonably  explained  as  the  result  of  tissue  dis- 
integration rather  than  renal  retention  and  indicate  that  the  renal 
changes  are  the  result  rather  than  the  cause  of  the  intoxication. 
Losee  and  Van  Slyke  could  find  no  increase  of  amino-acids  or  other 
intermediates  of  protein  destruction  in  either  blood  or  urine  in  preg- 
nancy toxemias,^"  their  total  nonprotein  blood  nitrogen  figures  ranging 
from  25  to  46  mg.  Similar  results  are  reported  by  Slemons,^^  who 
also  found  normal  amounts  of  fat,  with  increased  cholesterol  and 
decreased  lecithin,  and  after  the  convulsions  some  increase  in  blood 
sugar.  The  uric  acid  content  of  the  blood  is  high  (5-9  mg.).^-  With 
the  observed  low  blood  urea  of  eclampsia  it  is  difficult  to  account  for 
Hammett's^^  finding  of  a  high  urea  content  in  eclamptic  placentas. 
Macallum  describes  a  high  proportion  of  potassium  in  the  blood  in 
eclampsia. ^^ 

The  decrease  in  the  alkalinity  of  the  blood  observed  by  Zangmcister 
and  others  has  been  ascribed  to  the  formation  of  sarcolactic  acid  by 
Zweifel,**^  who  failed,  however,  to  find  an  excess  of  COo,  or  to  detect 
oxybutyric  acid  or  oxalic  acid  in  the  blood.  As  to  the  blood  pro- 
s'See  Zweifel  and  Lockmann,  Mlinch.  med.  Woch.,  1906  (53),  297;  Cent.  f. 
Gyn.,  1909  (33),  847. 

"  Zinsser,  Zeit.  f.  Geb.,  1912  (70),  200. 

f-*  Cent.  f.  Gyn.,  1910  (34),  6. 
'     s»  Amer.  Jour.  Med.  Sci.,  1914  (147),  .550. 

«"  Amer.  Jour.  Med.  Sci.,  1917  (153),  94,  corroborated  by  Morse,  Bull.  Johns 
Hopkins  Hosp.,  1917  (28),  199. 

"i  Amer.  Jour.  Obst.,  1918  (77),  717. 

82  Slcmonsand  Bogert,  Jour.  Biol.  Choni.,  1917  (32),  03. 

"Jour.  Biol.  Choiii.,  191S  (34),  515. 

•^Arner.  Jour.  Med.  Sci.,  1918  (150),  1. 

6»  Arch.  f.  Gyn.,  1905  (76),  537. 


PUERPERAL  ECLAMPSIA  543 

teins,  fibrin  ferment  has  been  found  increased  by  Dienst/^  while 
Schmidt  found  a  rehitive  increase  in  the  globuHn.  Sikes  concludes 
that  the  statements  to  be  found  in  the  hlciature  concerning  the  tox- 
icity of  the  blood  in  eclampsia  leave  notliing  pnjvcd  concerning  this 
point,  but  more  recent  studies  by  Graf  and  Landsteiner*''  affirm  an 
increase  of  toxicity  of  the  blood,  not  due  to  any  special  poison  but  to 
an  increase  in  the  amount  of  the  toxic  substances  ordinarily  present; 
however,  any  studies  of  toxicity  of  the  blood  are  of  doubtful  value 
because  of  the  reactions  produced  by  injections  of  even  normal  blood. 
More  attention  may  be  given  to  the  observation  of  Hiissey^^  that 
while  the  serum  of  pregnant  women  has  a  shght  vasodilator  effect,  in 
eclampsia  and  other  pregnancy  toxicoses  there  is  a  marked  vasocon- 
strictor effect.  The  antitryptic  titer  of  the  blood  may  be  much  in- 
creased.^^ Zangmeister^"  ascribes  impoTtance  to  edema  of  the  brain. 
Ballerini^^  found  that  the  physico-chemical  changes  in  the  blood  are 
quite  the  same  as  in  corresponding  conditions  of  nephritis.  An  in- 
crease in  the  sugar  content  of  the  blood  has  been  observed"^  but  no 
other  abnormality  of  carbohydrate  metabolism  is  usually  present. 
Blood  lipase  is  much  increased  because  of  the  hepatic  injury  (Whip- 
ple)." 

Theories  as  to  Etiology. — The  anatomical  changes  of  eclampsia 
are  such  as  to  leave  little  or  no  room  for  doubt  that  there  is  a  severe 
intoxication  with  poisons  that  have  a  markedly  toxic  effect  upon  all 
the  organs  of  the  body,  thus  differing  from  the  toxic  materials  at  work 
in  uremia,  which  seem  to  affect  chiefly  the  central  nervous  system  and 
to  produce  no  marked  tissue  changes.  Repeated  bacteriological  and 
histological  studies  have  failed  to  demonstrate  that  infection  with  either 
vegetable  or  animal  parasites  is  the  cause,  and  clinical  observations  do 
not  support  such  an  hypothesis.  The  association  of  the  condition  with 
pregnancy,  and  particularly  the  rapid  improvement  that  often  follows 
the  removal  of  the  contents  of  the  uterus,  almost  compels  us  to  admit 
that  the  causative  agent  is  produced  by  the  fetus  or  the  placenta. 
Some  investigators  (Politi,  Liepmann)  believe  that  they  have  found  a 
greater  degree  of  toxicity  in  extracts  from  the  placentas  from  eclamptic 
than  from  normal  women.  We  have  no  exact  ideas  as  to  the  nature 
of  the  supposed  toxic  substances,  except  that  recent  developments  in 
the  study  of  immunity  reactions  point  to  their  origin  from  proteolysis 
of  tissue  proteins,  presumably  from  the  placenta.     The  hypothesis  of 

6"  Arch.  f.  Gyn.,  1912  (96),  43;  Zeit.  Geb.  u.  Gvn.,  1919  (82),  102. 

"  Cent.  f.  Gvn.,  1900  (33),  142. 

«8Corrbl.  ScW.  Aerzte,  1918  (48),  691. 

«9  Franz,  Arch.  f.  Gyn.,  1914  (102),  579;  Ecolle,  Arch.  Mens.  Obst.  Gvn.,  1917 
(6),  97. 

^0  Deut.  med.  Woch.,  1911  (37),  1879. 

'1  Annali  Ostet.  e.  Gin.,  1910  (32),  273. 

"  Benthin,  Monats.  Geb.  u.  Gvn.,  1913  (37),  305;  Ryser,  Deut.  Arch.  khn.  Med., 
1916  (118),  408;  Siemens,  loc.  cit.^'- 

"  Jour.  Med.  Res.,  1913  (24),  357. 


544  ABNORMALITIES  IN  METABOLISM 

Zweifel  that  lactic  acid  is  responsible  seems  untenable,  and  the  de- 
gree of  acidosis  present  is  not  sufficient  to  account  for  the  intoxication 
(Losee  and  Van  Slyke) . 

The  Placenta  as  a  Source  of  Intoxication.^ — Histologists  having  fre- 
quently observed  placental  cells  in  the  blood  and  vessels  of  eclamptic 
patients,  it  was  once  suggested  that  multiple  capillary  emholi  of  pla- 
cental cells,  detached  from  chorionic  villi  and  forced  into  the  pla- 
cental circulation,  cause  the  manifestations  of  the  disease;  this  theory 
is  entirely  inadequate,  however,  to  explain  all  the  features  of  eclamp- 
sia. Related  to  this  hypothesis  is  the  idea  that  the  placental  tissues, 
being  foreign  to  the  maternal  organism  in  so  far  as  they  are  derived 
from  the  ovum,  give  rise  to  the  production  of  antibodies  (synajtioly- 
sins)  by  the  mother,  which  are  toxic  for  pregnant  animals  (Ascoli), 
and  which  may  have  to  do  with  eclampsia  in  some  unknown  way. 
Rosenau  and  Anderson  found  that  guinea  pigs  could  be  made  anaphy- 
lactic to  guinea-pig  placenta,  showing  conclusively  that  the  placenta 
contains  proteins  foreign  to  the  mother.  Attempts  to  establish  the 
anaphylactic  nature  of  eclampsia  have,  like  so  many  other  theories, 
foundered  on  the  fact  of  the  characteristic  anatomy  of  this  disease, 
which  is  never  seen  in  anaphylaxis.'^*  The  studies  of  Abderhalden 
have  shown  that  the  blood  of  every  pregnant  female  animal  contains 
enzymes  which  have  a  specific  proteolytic  action,  and  so  the  possibility 
exists  that  abnormal  or  excessive  products  of  such  proteolysis,  or  a 
lack  of  adequate  defensive  digestive  action,  may  be  responsible  for 
the  toxemias  of  pregnancy.  Esch'^^  and  Franz^^  have,  indeed,  found 
evidence  of  the  presence  in  the  serum  and  urine  of  eclamptics,  of  sub- 
stances resembling  anaphylactic  poisons  in  their  action,  and  presum- 
ably derived  from  proteolysis  somewhere  in  the  body.  Franz  found 
that  if  the  poison  injures  the  kidneys  seriously  it  is  retained  in  the 
body,  the  urine  ceasing  to  be  toxic,  which  has,  presumably  a  relation 
to  the  toxicosis  of  eclampsia. '''' 

Liepmann^*  and  others  have  reported  the  finding  of  a  considerable 
degree  of  toxicity  in  eclamptic  placentas,  but  this  is  probably  related 
to  the  increased  autolysis  observed  in  eclamptic  placentas  by  Dr}'- 
fuss.'^^     Obata^°  found  no  great  difference  in  the  toxicity  of  eclamptic 

^*See  Fellander,  Zeit.  Geb.  u.  Gyn.,  1911  (68),  26;  Mosbacher,  Deut.  med. 
Woch.,  1911  (37),  1021.  Vertes  (Monat.  Geb.  u.  Gyn.,  1914  (40),  361,  466) 
states  that  animals  dying  from  anaphylaxis  may  show  typical  eclamptic  tissue 
changes,  which  is  not  in  accordance  with  the  observations  of  many  others. 

"  Mlinch.  med.  Woch.,  1912  (59),  461. 

"  Ibid.,  page  1702. 

"  Hull  and  Rhodenburg  (Amer.  Jour.  Obst.,  1914  (70),  919)  ascribe  impor- 
tance to  leucine  derived  from  proteolysis  of  the  placental  elements,  while  Kiutsi 
(Zeit.  Geb.  u.  Gyn.,  1912  (72),  576)  considers  the  nucleins  of  the  placenta  the 
toxic  agents,  both  statements  being  unconfirmed  and  imjjrobable. 

'«  Munch,  med.  Woch.,  1905  (52),  687  and  2484;  Boos,  Boston  Med.  and  Surg. 
Jour.,  1908  (158),  612. 

"  Biochem.  Zeit.,  1908  (7),  493. 

*"  Jour,  lumiuiiol.,  1919  (4),  111;  bibliography  on  etiology  of  eclampsia. 


PUERPERAL  ECLAMPSIA  545 

and  normal  placentas,  but  believes  that  in  eclampsia  there  is  a  reduced 
capacity  of  the  maternal  blood  to  neutralize  this  poison.  According 
to  Mohr  and  Heimann,**^  the  eclamptic  placenta  shows  a  great  decrease 
in  lecithin,  which  they  ascribe  to  the  increased  autolysis,  and  to  the 
hydrolyzed  lecithin  they  attribute  the  hemotoxic  effects.  On  the 
other  hand  Murray  and  Bienenfeld*''^  report  the  finding  of  an  increased 
amount  of  lipoids  in  eclamptic  placenta. ^'^ 

The  Fetus  as  a  Source  of  Intoxication. — A  reasonable  view  of  the 
cause  of  eclampsia  is  that  it  is  initiated  by  the  excessive  or  abnormal 
products  of  metabolism  thrown  into  the  blood  of  the  mother,  both  from 
the  fetus  and  from  her  own  overactive  tissues;  these  cause  injury  to  the 
kidneys,  leading  to  a  further  retention,  or  injure  the  liver  so  that 
the  normal  metabolic  processes  of  that  organ  (particularly  oxidation) 
cannot  be  carried  on;  or,  perhaps  more  often,  both  liver  and  kidney 
as  well  as  other  organs  are  injured.  In  this  way  a  vicious  circle 
might  be  established  and  rapidly  lead  to  an  overwhelming  of  the  ma- 
ternal system  with  toxic  products  derived  from  both  her  own  and  the 
fetal  tissues.  It  must  be  admitted,  however,  that  the  rapid  improve- 
ment that  so  often  follows  removal  of  the  products  of  conception 
indicates  strongly  that  the  poisonous  substances  arise  chiefly,  if  not 
exclusively,  in  the  fetus  or  the  placenta.  But,  as  Liepmann  points 
out,  the  child  shows  relatively  little  evidence  of  intoxication,  while, 
on  the  other  hand,  eclampsia  may  develop  after  dehvery  of  the  fetus, 
which  facts  speak  in  favor  of  the  place  of  the  origin  of  the  poison 
being  the  placenta  and  not  the  fetus,  and  death  of  the  fetus  seems  to 
have  no  effect  on  the  eclampsia.^'*  Especially  important  in  this  con- 
nection is  the  observation  of  cases  of  eclampsia  in  patients  with  a  hy- 
datid mole  and  no  fetus. ^^ 

The  Ductless  Glands  in  Eclampsia. — In  view  of  the  mystery  surrounding  the 
cause  and  effect  of  the  enlargement  of  the  thyroid  during  pregnancy,  it  is  not 
strange  that  the  suggestion  has  been  made  that  the  enlargement  is  for  the  purpose 
of  neutralizing  the  excessive  amounts  of  toxic  materials  in  the  maternal  blood, 
and  that  failure  of  this  enlargement  is  responsible  for  eclampsia.  In  support  of 
this  idea  Lange^^  states  that  absence  of  the  normal  thyroid  enlargement  is  usual  in 
eclampsia,  and  Fruhinsholz  and  Jeandelize*^  note  the  frequency  of  eclampsia  in 
myxedematous  women.  The  notable  influence  of  calcium  upon  convulsions,  and 
the  possible  deficiency  in  calcium  during  pregnancy,  has  led  to  the  suggestion 
that  this  may  be  responsible  for  eclampsia,^*  and,  since  the  parathyroids  are  re- 
s' Ibid.,  1912  (46),  367. 

82  Jour.  Obst.  and  Gyn.  Brit.  Empire,  1910  (18),  225:  Biochem.  Zeit.,  1912 
(43),  245. 

*^  The  hypothesis  of  Mohr  and  Freund  that  oleic  acid  from  the  eclamptic 
placenta  is  a  hemolytic  factor,  is  not  corroborated  by  Polano  (Zeit.  Geb.  u.  Gyn., 
1910  (65),  581). 

s^  See  Lichtenstein,  Zeit.  f.  Gyn.,  1912  (36),  1419. 
^  85  Hitschmann,  Cent.  f.  Gyn.,  1904  (28),  1089.     See  also  Gross  (Prager  med. 
Woch.,  1909  (34),  (365)  who  found  records  of  seven  cases  of  eclampsia  with  hyda- 
tid mole,  with  or  without  a  fetus. 

8«  Zeit.  f.  Geb.  u.  Gyn.,  1899  (40),  34. 

8-  Presse  M4d.,  1902  (10),  1023. 

88  See  Silvestri,  Gaz.  degli  Ospcd.,  1910  (31\  G89;  Mitchell,  Med.  Record, 
1910  (78),  906. 


546  ABNORMALITIES  IN  METABOLISM 

lated  to  calcium  metabolism,  that  they  are  concerned;'^  but  such  theories  fail  to 
explain  the  many  changes  other  than  the  convulsions,  and  have  not  been  accorded 
much  importance.  Kastle  and  Healy'"  consider  that  parturient  paresis  of  cattle, 
which  bears  some  resemblance  to  human  eclampsia,  is  caused  by  absorption  of 
toxic  substances  produced  in  the  formation  of  the  colostrum;  it  is  cured  by  dilating 
the  lacteal  ducts  by  oxygen  or  other  means.  This  observation  lends  support  to 
the  theory  advanced  by  Sellheim^i  that  human  eclampsia  is  of  mammary  gland 
origin. 

Pernicious  Vomiting  of  Pregnancy. — This  condition  is  insepara- 
bly associated  with  eclampsia  and  non-convulsive  toxemias  of  preg- 
nancy, there  being  transitional  and  border-line  cases  of  all  sorts.  In 
fatal  cases  of  pernicious  vomiting  anatomical  changes  resembling 
those  of  eclampsia  have  been  found,  and  albuminuria  and  icterus  are 
often  observed. ^^  The  chief  chemical  interest  in  these  cases  lies  in 
the  urinary  findings,  there  being  commonly  observed  a  relatively 
high  proportion  of  ammonia  and  undetermined  nitrogen  with  de- 
creased urea,  which  findings  have  been  considered  indicative  of  defec- 
tive oxidation  or  deaminization  (Ewing  and  Wolf)  and  of  prognostic 
and  diagnostic  significance  (Williams).  There  is  also  excretion  of 
acetone  bodies  and  other  evidence  of  more  or  less  acidosis.^*  Under- 
bill and  Rand^^  hold  that  the  urinary  changes  are  entirely  compatible 
with  those  which  can  be  produced  by  starvation  which  is  present,  of 
course,  in  pernicious  vomiting;  but  Ewing^^  contends  that  there  are 
other  underlying  factors  beyond  those  of  starvation. 

Summary. — Most  of  the  facts  at  hand  speak  against  the  idea  that 
one  definite  chemical  substance  is  responsible  for  the  anatomical 
changes  and  symptomatic  manifestations  of  eclampsia.  More  proba- 
bly there  are  present  not  only  the  poisonous  substances  that  initiate 
the  tissue  changes,  and  which  probably  originate  in  the  placenta 
itself  or  from  digestion  of  placenta  proteins  in  the  maternal  blood  or 
organs,  but  also  toxic  substances  that  accumulate  because  of  the  dis- 
organization of  the  liver  and  kidney  cells,  and  which  are  possibly 
similar  to  the  toxic  substances  most  prominent  in  uremia  and  in  acute 
yellow  atrophy,  for  eclampsia  seems  to  stand  intermediate  between 
these  two  diseases,  encroaching  upon  the  characteristics  of  each. 
Acid  intoxication,  which  undoubtedly  exists  to  a  greater  or  less  de- 
gree in  some  cases  of  eclampsia,  is  not  an  important  cause  of  the  clinical 
manifestations  of  the  disease.  The  finding  of  minute  quantities 
of  lactic  acid  in  the  blood,  urine,  and  in  the  cerebrospinal  fluid  (Fiith 
and  Lockemann)  is  not  of  great  significance,  for,  as  Wolf^*^  rightly 

89  Massaglia  and  Sparapani,  Arch.  ital.  Biol.,  1907  (48),  109.  . 

90  Jour.  Infec.  Dis.,  1912  (10),  22G. 
»i  Zent.  f.  Gyn.,  1909  (U),  1G09. 

92  See  Ewing  and  Wolf,  Amer.  Jour.  Obstr.,  1907  (55),  289. 

93  See  (lilliatt  and  Konnaway,  (Juart.  Jour.  Med.,  1919  (12),  61;  Losoo  and  Van 
Slyke,  Aincr.  Jour.  Med  Sci.,  1917  (153),  94;  Duncan  and  Harding,  Canad.  Mod. 
Assoc.  Jour.,  191 S  (S),  1057. 

•^  Arch.  Int.  iMed.,  1910  (5),  Gl. 

»»  Amer.  Jour.  Med.  Sci.,  1910  (139),  828. 

9«  New  York  Med.  Jour.,  1906  (83),  813. 


ACUTE  YELLOW  ATROPHY  OF  THE  LIVER  547 

insists,  similar  amounts  may  be  found  in  other  conditions  associated 
with  convulsions  and  partial  asphyxia,  or  in  partial  starvation,  such 
as  results  from  the  vomiting  of  pregnancy.  The  excretion  of  these 
organic  acids,  as  well  as  the  large  proportion  of  unoxidized  sulphur 
in  the  urine,  indicates  that  incomplete  oxidation  is  an  important 
feature  of  eclampsia,  and  under  such  conditions  a  large  number  of 
imperfectly  known  toxic  substances  may  accumulate  in  the  blood 
and  tissues.  The  defective  oxidation  indicated  by  the  urinary  find- 
ings arc  probablj^  the  result  of  the  injury  to  the  liver-cells,  wliich 
have  such  a  prominent  oxidizing  function.  The  hypotheses  which 
ascribe  the  intoxication  to  products  of  specific  proteolysis  of  the  for- 
eign proteins  of  the  placenta  which  have  entered  the  maternal  organ- 
ism, are  suggestive,  but  as  yet  are  not  sufficiently  developed  to  permit 
of  any  definite  conclusions  as  to  the  extent  to  which  they  apply. 

ACUTE  YELLOW  ATROPHY  OF  THE  LIVER 

In  this  condition  there  is  presented  a  striking  picture  of  autolysis, 
in  that  a  large  parenchymatous  organ  undergoes  a  rapid  reduction 
of  size  because  of  a  solution  of  its  structural  elements,  while  at  the 
same  time  products  of  protein  digestion  (leucine,  tyrosine,  etc.) 
appear  free  in  the  liver,  the  blood,  and  the  urine.  Because  of  these 
prominent  features  and  their  relation  to  the  questions  of  metabohsm 
in  general,  and  the  function  of  the  hver  in  particular,  acute  yellow 
atrophy  of  the  liver  has  been  the  object  of  much  greater  interest  and 
investigation  than  its  clinical  importance  would  warrant,  for  it  is 
not  a  common  disease. ^^ 

The  etiologj^  of  the  disease  is  quite  unknown,  but  it  is  very  probably 
not  a  specific  one  for  we  find  that  numerous  forms  of  intoxication 
may  lead  to  a  condition  closely  resembling  acute  yellow  atrophy,  .^^ 
particularly  puerperal  eclampsia,  and  some  cases  of  septicemia  (espe- 
cially with  the  streptococcus),^^  and  poisoning  with  phosphorus, 
arsenic,  nitrophenols  and  mushrooms.^  It  seems  probable  that  any 
poison  w^hich  does  not  directly  cause  death,  but  which  causes  a  severe 
injury  to  the  liver-cells  without  at  the  same  time  destroying  the  auto- 
lytic  enzymes,  so  that  the  cells  die  and  undergo  rapid  autolysis,  maj' 
produce  a  condition  identical  with  or  similar  to  acute  yellow  atrophy 
(Wells  and  Bassoe).-  Inthetypicalcasesof  the  disease,  of  "idiopathic" 
origin,  the  poisonous  agent  possibly  comes  from  the  alimentary  canal, 
as  indicated  by  a  prehminary  period  of  gastro-intestinal  disturbance 

'^  Up  to  1903  there  had  been  reported  about  500  cases  (Best,  Thesis,  University 
of  Chicago,  1903). 

'*  It  is  to  be  borne  in  mind  that  the  color  is  yellow  only  during  the  earlier 
stages,  "red  atrophy"  occurring  later,  but  the  name  acute  "yellow  atrophy"  has 
come  through  usage  to  apply  to  the  disease  as  a  whole. 
""  Babes,  Ann.  Inst.  Path.  Bucarest,  vol.  6. 

1  Frev,  Zeit.  klin.  Med.,  1912  (75),  455;  Prym,  Virchow's  Arch.,  1919  (226),  229. 
-  Jour.  Amer.  Med.  Assoc,  1904  (44),  685. 


548  ABNORMALITIES  IN  METABOLISM 

that  usually  precedes  the  onset  of  the  disease,  and  secondly  by  the 
fact  that  the  liver  seems  to  receive  the  chief  effect  of  the  poison. 
Whether  these  h3^pothetical  poisons  are  produced  by  abnormal  fer- 
mentation and  putrefaction  in  the  alimentary  tract,  or  by  a  specific 
organism  elaborating  its  poison  in  this  location,  is  quite  unknown. 
Bacteriological  studies  of  the  disease  have  so  far  given  inconstant  and 
non-instructive  results.  In  the  countries  where  phosphorus  poisoning 
is  common  (especially  Austria)  there  has  been  found  much  difficulty 
in  distinguishing  in  many  cases  the  results  of  phosphorus  poisoning 
from  acute  yellow  atrophy  of  the  fiver,  and  many  have  contended 
that  there  is  no  real  difference;  i.e.,  that  phosphorus,  as  well  as  un- 
known poisons,  may  cause  acute  yellow  atrophy.  The  present  trend 
of  opinion,  however,  seems  to  favor  the  view  that  there  is  a  primary 
liver  atrophy  which  is  different  from  that  caused  by  phosphorus  or 
other  known  poisons  in  several  essential  respects.^ 

Phosphorus  Poisoning. — Between  phosphorus  poisoning  and  "primary"  hepatic 
atrophy  the  following  chief  differences  may  he  discerned:  Phosphorus  produces  a 
general  injurious  effect  upon  all  the  organs  of  the  body,  the  liver  merely  showing 
the  most  marked  anatomical  changes,  which  at  first  consist  of  a  fatty  metamor- 
phosis of  the  liver,  due  to  migration  of  the  body  fat  from  the  fat  deposits  into  the 
injured  cells  ,(Roseneld,  Taylor);  subsequently  the  liver  cells  disintegrate, 
the  cytoplasm  being  affected  before  the  nucleus,  and  the  liver  may  be- 
come smaller  than  normal,  although  it  is  usually  enlarged  because  of  the  fat 
deposition.  Typical  acute  yellow  atrophy  is  characterized  by  an  early  necrosis 
of  a  large  proportion  of  the  liver-cells,  the  nucleus  becoming  unstainable  while  the 
cytoplasm  is  still  little  altered  in  appearance,  and  fatty  changes  play  a  subordi- 
nate role  or  are  absent.  As  Anschtitz  says,  the  poison'seems  to  strike  at  the  life 
of  the  cell,  its  nucleus,  while  phosphorus  attacks  the  cytoplasm.  Furthermore, 
the  poison  of  yellow  atrophy  seems  to  be  very  specific,  for  it  attacks  the  other 
organs  of  the  body  almost  not  at  all,  and  within  the  liver  it  affects  only  the  hepatic 
cells  proper,  while  the  bile-duct  epithelium  and  the  stroma  cells  are  so  little  injured 
that  they  are  able  to  proliferate  greatly,  this  proliferation  being  a  prominent  feature. 
There  are  also  clinical  and  chemical  differences  that  will  be  discussed  later,  but 
yet,  on  the  whole,  the  resemblances  of  yellow  atrophy  and  phosphorus  poisoning 
are  so  great  that  we  have  obtained  much  information  concerning  the  former  by 
means  of  experimental  studies  of  phoshorus  poisoning. 

Delayed  Chloroform  Poisoning. — After  chloroform  narcosis,  and  rarely  after 
ether,  there  occasionally  develops  a  severe  intoxication,  with  clinical  and  anatomical 
findings  very  similar  to  acute  yellow  atrophy  and  i)hosphorus  poisoning;'  in  point 
of  the  fatty  changes  the  cases  usually  stand  intermediate  between  acute  yellow 
atrophy  and  phosphorus  poisoning.  This  action  of  chloroform  would  seem,  from 
the  studies  of  Evarts  Graham,^  to  be  produced  l)y  the  liydrochloric  acid  formed 
from  it  in  the  liver.     Some  cases  of  puerperal  eclampsia  also  present  such  profound 

2  See  Anschlitz,  Arb.  a.  d.  Path.  Inst.  Tubingen,  1902  (3),  280;  Paltauf,  Verh. 
Deut.  Path.  Gesell.,  1903  (5),  91;  Riess,  Berl.  klin.  Woch.,  1905  (42),  No.  4-la, 
p.  54. 

••Complete  review  and  literature  by  Bevan  and  Favill,  Jour.  Amer.  Med. 
Assoc,  1905  (45),  691;  Muskens,  Mitt.  Grenz.  Med.  u.  Cliir.,  1911  (22),  56S.  Full 
discussion  of  chemistry  of  chloroform  necrosis  liy  ^^'olls,  Jour.  Biol.  Cliom.,  190S 
(5),  129.  Exixniinental  necrosis — see  \\'lui)ple  and  Sporrv,  Johns  Hopkins 
Hosp.  Bull.,  1909  (20),  278;  Graham,  Jour.  Exper.  Med.,  1912  (15),  307;  Simonds, 
Arch.  Int.  Med.,  1919  (23),  3()2;  Davis  and  Whipple,  ibid.,  p.  012. 

'■  Jour.  Ex)).  Med.,  1915  (22),  48.  Tliis  hypothesis  receives  supi)ort  from  the 
conclusion  reached  by  many  investigators  that  ilichl()rethylsuli)hide  ("mustard 
gas")  also  injures  tissues  through  intracellular  dissociation  liberating  IICI.  (See 
Lillie  ('/  ai,  Jour.  Pharm.,  1919  (14),  75.) 


ACUTE  YELLOW  ATROPHY  OF  THE  LIVER  549 

liver  changes  that  they  are  distingui.shed  as  echinipsia  chiefly  on  the  basis  of  the 
convulsive  manifestations,  rather  than  on  the  ground  of  anatomical  changes.  So, 
too,  the  hepatic  changes  in  certain  septicemias  and  acute  syphilis  may  resemble 
those  of  acute  yellow  atrophy  to  a  greater  or  less  degree. 

Summary  of  Views  on  Etiology. — From  a  review  of  the  literature 
and  the  study  of  a  few  cases,  the  writer  has  reached  the  following 
imderstanding  of  the  condition  described  as  acute  yellow  atrophy  of 
the  liver:  The  "atrophy"  is  due  entirely  to  autolysis  of  necrotic 
liver-cells  by  their  own  enzymes.  In  the  most  typical  cases  of  "pri- 
mary" or  "idiopathic"  yellow  atrophy  we  have  to  do  with  a  poison 
having  a  very  specific  effect  on  the  liver-cells,  which  destroys  their 
"life"  {i.  e.,  stops  synthetic  activities)  without  injuring  their  intra- 
cellular proteolytic  enzymes,'  and  consequently  autolj'sis  occurs;  as 
the  poison  affects  other  organs  but  little,  the  necrosis  and  autolysis 
continue  until  there  is  so  much  loss  of  liver  function  that  systemic 
poisoning  results  from  the  hepatic  insufficiency  and  from  the  resulting 
accumulation  of  poisonous  products  of  incomplete  metabolism.  That 
the  intoxication  comes  in  large  measure  from  the  changes  in  the  liver, 
even  in  phosphorus  poisoning,  is  shown  by  the  greater  resistance  to 
phosphorus  of  dogs  with  Eck's  fistulas.^  The  patient  dies  from  this 
poisoning,^  and  the  liver  is  found  at  autopsy  to  have  decreased  by 
from  one-third  to  one-half  or  more  in  its  volume.  This  great  change 
would  not  be  possible  if  the  poisons  affected  the  heart,  kidneys,  or 
brain  as  much  as  they  do  the  liver  structure,  which  is  probably  the 
reason  that  phosphorus,  bacterial  poisons,  snake  poisons,  and  other 
poisons  that  affect  many  sorts  of  cells  do  not  ordinarily  produce  the 
typical  picture  of  liver  atrophy.  When  these  poisons  affect  the  liver 
more  and  the  other  tissues  less,  we  approach  the  condition  of  acute 
yellow  atrophy;  e.  g.,  if  the  dose  of  phosphorus  is  not  so  great  as  to 
kill  the  patient  through  injury  of  other  more  vital  organs,  after  a  few 
days  the  necrosed  liver-cells  undergo  autolysis,  and  if  enough  liver- 
cells  have  been  destro3''ed,  hepatic  insufficiency  may  cause  death, 
with  the  finding  of  an  anatomical  condition  in  the  liver  that  can  be 
properly  designated  as  acute  atrophy.  Hence  it  is  possible  for  many 
poisons  to  cause  this  condition  under  certain  circumstances,  and  there 
seem  to  be  certain  unknown  poisons  (possibly  of  intestinal  origin^) 
that  are  of  such  a  nature  that  thej^  cause  specifically  acute  hepatic 
atrophJ^  The  above  hypothesis  seems  to  explain  all  the  known  facts 
concerning  this  disease.  That  phosphorus,  chloroform,  and  some 
other  poisons  lead  particularly  to  fatty  changes  may,  perhaps,  be  due 

^  According  to  some  investigators  phosphorus  augments  autolysis  even  in  vitro 
(see  Krontowski,  Zeit.  f.  Biol.,  1910  (54),  479). 

'  Fischler  and  Bardach,  Zeit.  physiol.  Chem.,  1912  (78),  435. 

8  The  mortality  of  cases  sufhciently  typical  to  be  diagnosed  antemortem  is 
estimated  by  Rondaky  (Roussky  Vratch,  Oct.  28,  1900)  at  97  to  98  per  cent. 
Concerning  the  regenerative  changes  in  the  cases  which  recover,  see  Yamasaki 
(Zeit.  f.  Heilk.,  Path.  Abt.,  1903  (24),  248). 

«  See  Carbone,  Riforma  Med.,  1902  (1),  687  and  698. 


550  ABNORMALITIES  IN  MET  A  BOLISM 

to  their  acting  especially  upon  the  oxidizing  enzymes/"  leaving  the 
autolytic  enzymes  and  the  lipase  free  to  digest  the  cell  and  to  form 
fat.^^  That  it  is  particularly  the  oxidizing  enzymes  that  are  attacked 
is  well  shown  by  the  chemical  findings,  and  also  by  Loewy's^^  observa- 
tion that  in  poisoning  with  CNH,  which  acts  by  impairing  oxidation, 
the  alterations  in  protein  metabolism  are  very  similar  to  those  of 
phosphorus  poisoning.^*  To  be  sure,  Lusk^^  found  no  deficiency  in 
general  oxidation  in  phosphorus  poisoning,  but  this  does  not  signify 
that  the  local  changes  do  not  depend  upon  local  defective  oxidative 
processes.  Furthermore,  the  marked  power  of  sugar  to  protect 
the  liver  from  such  poisons  as  phosphorus  and  chloroform  seems  to 
depend  on  its  furnishing  easily  oxidizable  material  to  cells  with  reduced 
oxidative  capacity  (Simonds).^^ 

Not  only  phosphorus  but  many  metals,  especially  mercurj^,  seem 
able  to  cause  the  anatomical  changes  of  acute  yellow  atrophj-,  for 
the  condition  has  been  observed  very  frequently  in  persons  receiving 
mercurial  and  arsenical  treatment  for  syphilis.  ^^  Here  the  syphilis 
has  been  held  responsible  by  some,  but  the  fact  that  in  many  of  the 
cases  the  syphilis  was  quiescent  or  chronic  at  the  time,  and  that  mer- 
cury and  arsenic  are  known  to  kill  cells  and  stimulate  autolysis,  seems 
to  incriminate  the  metals, ^'^  at  least  in  some  cases.  On  the  other  hand, 
Stewart, ^^'^  calls  attention  to  the  fact  that  acute  yellow  atrophy  occurs 
especially  often  after  poisoning  with  picric  acid,  trinitr o-toluene,  dini  tro- 
benzene  and  aromatic  arsenicals;  as  in  all  these  substances  the  only 
common  component  is  the  benzene  radical,  its  responsibility  is  strongly 
suggested. 

Chemical  Changes  of  Acute  Yellow  Atrophy 

The  Urine. — Most  striking,  and  long  regarded  as  pathognomonic, 
is  the  presence  of  leucine  and  tyrosine  in  the  urine,  first  described  by 

loSee  Verworn,  Deut.  med.  Woch.,  1909  (35),  1593;  Joannovics  and  Pick» 
Arch.  ges.  Physiol.,  1911  (140),  327. 

11  Wells,  Jour.  Amer.  Med.  Assoc,  1906  (46),  341. 

12  Cent.  f.  Physiol.,  1906  (19),  23. 

1^  The  hypothesis  suggested  by  Quincke  (Nothnagel's  Handbook,  1899,  vol.  18, 
p.  307)  that  possibly  regurgitation  of  pancreatic  juice  up  the  bile  ducts  might 
be  responsible  for  the  degenerative  changes  in  the  liver,  is  contradicted  by  the 
fact  that  the  bile  pressure  is  greater  than  the  pancreatic  juice  pressure,  and  that 
the  bile-ducts  and  peripheral  portions  of  the  lobules  are  least  affected.  Nor 
could  Best""  prove  that  trypsin  injected  into  the  liver  by  way  of  the  bile-ducts  is 
able  to  cause  such  changes.     (See  Wells  and  Bassoe.^) 

'■*  Science  of  Nutrition,  Philadelphia,  1909. 

"  Arch.  Int.  Med.,  1919  (23j,  362.  P>vin,  however,  ascribes  the  protective 
effect  of  carbohydrate  feeding  to  the  glycogen,  which  protects  the  protein-fat 
emulsion  of  the  liver  cell  cytoplasm  from  the  action  of  acids.  (Jour.  Lab.  Clin 
Med.,  1919  (5),  146). 

i«  Severin,  Zeit.  klin.  Med.,  1912  (76;,  138.  Bendig,  Mlinch.  med.  Woch.,  1915 
(62),  1144. 

"  Tilcston  (Boston  Med.  and  Surg.  Jour.,  1908  (158),  510)  has  described  a 
case  of  acute  yellow  atrophy  from  mercurialisin  without  .svphilis. 

""Stewart,  Vining  and'Bibby,  Jour.  Path.  Pact.,  1919  (.23),  Proc.  Path.  See, 
p.  120. 


ACUTE  YELLOW  ATROPHY  OF  THE  LIVER  551 

Frerichs.  While  we  now  know  that  these  and  other  amino-acids 
may  occur  in  the  urine  in  any  conditions  in  which  there  is  a  great 
breaking  down  of  tissue  within  the  body,  yet  it  is  true  that  in  no 
other  con(Ution  are  they  found  so  abunchmtly  as  in  acute  hepatic 
atrophy  (as  high  as  1.5  gm.  of  tyrosine  per  diem  has  been  found). ^' 
They  are  nearly  constantly  present  (in  thirteen  out  of  fourteen  cases 
studied  by  Riess),^^  tyrosine  being  usually  the  more  abundant.  Deu- 
tero-proteose  is  also  frequently  (but  not  constantly)  found,  as  further 
evidence  of  abnormal  protein  splitting.-"  Uric  acid  and  other  purines 
are  often  somewhat,  but  not  characteristically,  increased,  probably 
resulting  from  the  nuclear  destruction  in  the  liver.  There  is  often  an 
increase  in  ethereal  sulphates  (Salkowski),^^  and  in  phosphorus  poi- 
soning various  bases  have  been  found  in  the  urine,--  which  presumably 
might  also  be  found  in  acute  yellow  atrophy  if  sought  for.  The  total 
elimination  of  nitrogen  is  increased-^  (particularly  if  the  scanty  intake 
is  considered),  and  the  proportion  that  appears  as  urea  is  decreased, 
largely  because  of  the  pi'esence  of  much  ammonia,-^  part  of  which,  at 
least,  is  eliminated  combined  with  organic  acids.  Chief  of  these  acids 
is  sarcolactic  acid,  but  of  particular  interest  is  the  supposed  appearance 
of  oxijmandelic  acid, 

H0<^    ^CHOH— COOH, 

which  might  be  derived  from  tyrosine  (Schultzen  and  Riess), 

ho/    ^CH2— CH(NH2)— COOH, 

by  the  splitting  out  of  the  NH2  group,  the  benzene  nucleus  failing 
to  be  completely  oxidized  as  it  normally  is.  The  researches  of  El- 
linger  and  Kotake,^^  however,  make  it  probable  that  the  supposed 
oxymandelic  acid  is  something  else,  most  likely  p-oxyphenijl-ladic 
acid, 

ho/  \ch2— choh— cooh 

1*  An  interesting  exception  has  been  reported  by  W.  G.  Smith  (Practitioner, 
1903  (70),  155)  who  found  great  quantities  of  leucine  in  the  urine  of  a  young 
woman  who  was  apparently  not  at  all  ill.  Rosenbloom  has  found  tyrosine  crys- 
tals in  the  urine  of  a  healthy  pregnant  woman,  and  cites  other  cases  of  tyrosin- 
uria  without  hepatic  atrophy  (N.  Y.  Med.  Jour.,  Sept.  19,  191-4). 

'^  Berl.  khn.  Woch.,  1905  (42),  No.  44  a.,  p.  54. 

-">  Salkowski  (Berl.  klin.  Woch.,  1905  (42),  1581)  found  in  the  urine  of  a  case 
of  acute  3'ellow  atroph}^  a  large  quantity'  of  nitrogen  iii  a  colloidal  but  non-protein 
form,  apparently  of  carbohydrate  naWre.  Mancini  (i\jch.  di  farm,  sperim., 
190G,  Bd.  v)  also  observed  an  increase  in  the  colloidal  nitrogen  of  the  urine  in  liver 

21  Virchow's  Arch.,  1909  (198),  188. 

"  Takeda,  Pfliiger's  Arch.,  1910  (133),  365. 

23  See  Welsch,  Arch.  int.  pharm.  et  ther.,  1905  (14),  211. 

2*  See  Voegtlin,  Johns  Hopkins  Hosp.  Bull.,  1908  (19),  50;  Wliite,  Boston 
Med.  and  Surg.  Jour.,  1908  (158),  729. 

"  Zeit.  physiol.  Chem.,  1910  (65),  397  and  402;  also  Fromherz,  ibid.,  1911  (70), 
351. 


552  ABNORMALITIES  IN  METABOLISM 

which  can  be  demonstrated  in  the  urine  of  dogs  poisoned  with  phos- 
phorus, and  which  represents  a  simple  deaminization  of  tyrosine  without 
further  oxidation.  It  is  evident  from  the  urinary  findings,  therefore, 
that  oxidation  is  decreased,  which  is  presumably  because  of  the  de- 
struction of  liver  tissue  with  its  important  oxidizing  functions.  The 
reduction  of  oxidation  can  also  be  shown  experimentally  by  studying 
the  respiratory  exchange,  Welsch  having  found  the  oxidation  decreased 
by  from  ^  to  }i  in  phosphorus  poisoning.  Carbamates  do  not  seem  to 
be  present  in  recognizable  amounts,  and  sugar  is  also  absent. 

In  phosphorus  poisoning  the  urinary  findings  are  similar,  but  with  marked 
quantitative  differences.  Tyrosine  cannot  usually  be  detected,  at  least  by  ordinary 
methods,  being  found  by  Riess  in  but  7  of  36  cases  of  (human)  phosphonis  poison- 
ing, and  in  but  4  of  these  was  it  abvmdant.  Leucine  is  even  less  frequently  found. 
With  experimental  animals  glycine  and  other  amino-acids  have  been  found"  in  the 
urine,  and  they  could  probably  be  found  in  acute  hepatic  atrophy  if  the  same 
delicate  methods  were  employed.  Wohlgemuth'^  has  indeed  found  glycine, 
alanine,  and  arginine  in  human  urine  after  phosphorus  poisoning.  The  small 
quantity  of  amino-acids  in  phosphorous  poisoning  is  probably  due  to  the  relative 
slowness  of  the  autolytic  changes.  On  the  other  hand,  the  deficiency  of  oxidation 
in  phosphorus  poisoning  is  shown  by  the  abundant  elimination  of  organic  acids, 
Riess  having  obtained  as  high  as  4  to  6  grams  of  the  zinc  salt  of  paraladic  acid 
from  the  urine  (per  liter)  in  human  cases,  and  its  presence  seems  to  be  constant. 

The  Liver.2^ — In  the  liver  may  be  found  an  abundance  of  the  free 
amino-acids  that  have  not  yet  escaped  by  diffusion,  their  presence 
having  been  first  detected  by  Frerichs  microscopically.  Taylor-^  was 
able  to  isolate  from  a  liver  weighing  900  grams,  0.35  gm.  of  leucine 
and  0.612  gm.  aspartic  acid,  which  probably  represent  much  less  than 
the  total  amount  present.  Deuteroalbumose  was  also  found,  but  no 
peptone,  arginine,  histidine,  or  lysine,  and  glycogen  was  also  absent. 
In  another  case  that  appeared  to  be  the  result  of  chloroform  intoxi- 
cation, Taylor^"  obtained  4  grams  of  leucine,  2.2  grams  of  tyrosine, 
and  2.8  grams  of  arginine  nitrate.  Wells  found  several  amino-acids 
free  in  sufficient  quantity  to  identify  in  the  liver  in  cases  of  acute  yellow 
atrophy  and  chloroform  necrosis,  an  increase  in  gelatigenous  substance 
in  the  former,  and  of  organic  non-lipoidal  phosphorus  in  both,  sulphur 
being  unchanged.  The  increase  in  tissue  phosphorus  is  striking,  and 
agrees  with  Slowtzoff's  and  Wohlgemuth's^^  finding  that  the  tissue 
phosphorus  persists  in  experimental  phosphorus  poisoning.  Wake- 
man^2  found  that  in  phosphorus  poisoning  of  dogs  the  liver  shows 
a  diminution  of  the  hexonc  bases  as  a  whole,  the  arginine  being  espe- 

28  Abderhalden  and  Barker,  Zeit.  physiol.  Chem.,  1904  (42),  524;  Abderhalden 
and  Bergell,  ibid.,  1903  (39),  464. 

"  Zeit.  physiol.  Chem.,  1905  (44),  74. 

"  Full  analyses  and  discussion  of  the  chemistry  of  the  liver  in  acute  >ellow 
atrophy  and  choloroform  necrosis  given  by  Wells,  Jour.  lOxper.  Med.,  1907  (9), 
027;  Arch.  int.  Med.,  1908  (1),  5S9;  Jour.  Biol.  Cliem.,  190S  (5),  129. 

2»  Zeit.  physiol.  Cliem.,  1902  (34),  5S0;  Jour.  Mod.  Ucscarcli,  1902  (S),  424. 

3"  Univ.  of  Calif.  Publications  (Pathol.),  1904  (1),  43. 

3>  Biocheiii.  Zeit.,  1911  (32),  172. 

3^  Jour.  Expcr.  Mod.,  1905  (7),  292;  Jour.  Biol.  Chem.,  1908  (4),  119. 


ACUTE  YELLOW  ATROPHY  OF  THE  LIVER  553 

cially  reduced;  but  no  such  change  was  found  by  him  in  acute  yellow 
atrophy,  nor  by  Wells  in  chloroform  necrosis.  Jackson  and  Pearce'^ 
found  an  increase  in  the  diamino  nitrogen  with  extensive  necrosis  of 
the  liver  in  dogs  and  horses.  Wohlgemuth'^'*  found  arginine  in  the  urine 
in  phosphorus  poisoning.  The  lecithin  of  the  liver  is  also  decreased 
(Heffter'^  and  Wells),  and  the  increase  in  P2O6  observed  in  the  urine 
presumably  comes  partly  from  this  source;  cholesterol  is  unchanged. 
Beebe^*^  found  the  pentose  of  the  liver  not  greatly  altered  from  the 
normal  relations.  The  typical  idiopathic  atrophied  liver  shows  little 
or  no  increase  in  fat,  either  chemically  or  microscopically,  whereas  there 
is  considerable  replacement  of  the  lost  liver  substance  by  water,  as 
shown  in  the  following  table: 

Fat-free 
Dried 
Water  Fat  Substance 

Normal  liver  (Quincke) 76.1  3.0  20.9 

Normal  liver  (Wells) 77.6  5.0  17.4 

Acute  atrophv  (Perls) 81.6  8.7  9.7 

Acute  at rophv  (Perls) 76.9  7.6  15.5 

Acute  atrophv  (v.  Starck) 80 . 5  4.2  15.5 

Acute  atrophy  (Taylor) 85 . 8  2.0  12.2 

Acute  atrophy  (Wakeman) 79.3  ...  .... 

Acute  atrophy  (Wells) 83 . 8  2.5  13.7 

Acute  atrophy  (Voegtlin) 78 . 0  6.6  15.4 

Phosphorus  poisoning  (v.  Starck) 60.0  29.8  10.0 

Fatty  degeneration  (v.  Starck) 64 . 0  25 . 0  11.0 

Chloroform  necrosis  (Wells) 72 . 4  8.8  18.8 

Similar  results  have  been  obtained  frequently  by  other  observers, 
Taylor  estimating  that  in  his  case  about  three-fourths  of  the  liver 
parenchyma  had  disappeared.  The  yellow  color  of  the  liver  tissue 
characteristic  of  this  condition  seems  to  be  due  to  bilirubin  rather 
than  to  fat,  because  as  soon  as  the  tissues  are  put  into  oxidizing  agents 
(e.  g.,  dichromate  hardening  fluids)  they  turn  grass-green  from  the  oxi- 
dation of  the  bilirubin  into  biliverdin.  There  seems  to  be  a  marked 
increase  in  free  fatty  acids,  probably  the  unsaturated  higher  fatty 
acids,  which  are  strongly  hemolytic.^''  Glycogen  is  greatly  reduced 
in  phosphorous  and  chloroform  poisoning,  and  presumably  in  acute 
yellow  atrophy. 

Jacoby^^  found  that  the  livers  from  phosphorus-poisoned  dogs 
underwent  autolysis  with  greater  rapidity  than  normal  livers,  which 
was.  attributed  to  increased  activity  or  amount  of  the  autolytic  en- 
zymes, although  addition  of  phosphorus  to  a  solution  containing  liver 
ferments  was  not  found  to  increase  their  activity.  The  aldehydase 
w^as  not  found  decreased,  and  tyrosinase  could  not  be  demonstrated, 

"  Jour.  Exper.  Med.,  1907  (9),  520. 
3<  Zeit.  phvsiol.  Chem.,  1905  (44),  74. 
3^  Arch.  exp.  Path.  u.  Pharm.,  1891  (28),  97. 
36  Amer.  Jour,  of  Phvsiol.,  1905  (14),  237. 
"  Joannovics  and  Pick,  Berl.  klin.  Woch.,  1910  (47),  928. 

38  Zeit.  physiol.  Chem.,  1900  (30),  174;  see  also  Porges  and  Pribram,  Arch, 
e.xp.  Path.  u.  Pharm.,  1908  (59),  20. 


554  ABNORMALITIES  IN  METABOLISM 

but  Slowtzoff^^  found  both  peroxidase  and  protease  decreased,  and 
attributed  the  increased  autolysis  to  a  greater  acidity  of  the  Uver. 
Burge*"  describes  decrease  in  the  catalase  of  hver  and  blood  in  experi- 
mental phosphorus  poisoning,  and  Simonds^^  found  hepatic  ereptase 
also  decreased,  but  not  in  chloroform  poisoning;  esterase  was  not 
altered. 

The  Blood. — In  the  blood  marked  changes  are  found,  one  of  the 
most  prominent,  besides  the  icterus,  being  the  decreased  coagulability 
of  the  blood.  This  seems  due  to  a  loss  of  fibrinogen,"*-  which,  with 
the  globulin,  is  greatly  decreased,  the  albumin  remaining  less  altered. ^^ 
The  fibrin-ferment  also  seems  to  be  decreased.  These  changes  may 
be  due  to  direct  autolysis  of  the  blood  constituents  (Jacoby  having 
found  that  thrombi  become  rapidly  dissolved  in  phosphorus-poison- 
ing) or  to  the  changes  in  the  liver.  The  icterus  depends  apparently 
upon  lesions  of  the  finest  bile  capillaries,*^  although  there  is  also  some 
increase  in  hemolysis,  and  a  decrease  in  the  total  blood  and  all  its 
elements  (Welsch);*'  and  both  bile  salts  and  pigments  appear  in  the 
urine.  In  all  these  diseases  with  marked  liver  changes  there  is  an 
increase  in  the  lipase  of  the  blood. ^"^  Neuberg  and  Richter'*^  have 
analyzed  the  blood  drawn  during  life  from  a  patient  with  acute  hepatic 
atrophy,  and  isolated  from  355  c.c.  of  blood  0.787  gm.  tyrosine,  1.102 
gm.  leucine,  and  0.240  gm.  of  lysine;  they  estimated  the  amount  of 
free  amino-acids  in  the  entire  blood  to  be  about  30  grams. *^  This 
amount  is  so  large  that  they  question  the  possibilit}'  of  it  all  arising 
from  the  degenerated  liver  tissue.  In  dogs  suffering  from  chloroform 
necrosis  of  the  liver  or  phosphorus  poisoning  the  amount  of  free  amino 
acids  in  the  blood  and  urine  is  usually  very  small. "'^ 

By  the  use  of  Van  Slyke's  method  it  has  been  found  that  acute 
yellow  atrophy  is  accompanied  by  the  highest  amino-N  figures  in 
the  blood  recorded  in  any  disease,  Feigl  and  Luce^"  having  reported 

39Biochem.  Zeit.,  1911  (31),  227. 

"  Amer.  Jour.  Physiol.,  1917  (43),  545. 

*i  Jour.  Exp.  Med.,  1918  (28),  673. 

«  Whipple  and  Hunvitz  (Jour.  Exper.  Med.,  1911  (13),  136)  find  a  great 
decrease  in  fibrinogen  during  experimental  choloroform  necrosis  of  the  liver. 

*^  Jacoby,  loc.  cit.;^^  see  also  Doj'on,  Compt.  Rend.  Soc.  Biol.,  1905  (58),  493; 
and  1909,  Vol.  66. 

/^  Lang  (Zeit.  exp.  Path.,  1906  (3),  473)  found  fibrinogen  in  the  bile  of  a  dog 
poisoned  with  phosphorus,  which  may  account  for  the  occlusion  of  the  bile  vessels 
and  the  resulting  jaundice. 

«  Arch.  int.  Pharm.  et  Th6r.,  1905  (14),  197. 

*«  Whipple  et  al..  Bull.  Johns  Hopkins  Hosp.,  1913  (24),  207  and  357.  Quinan 
found  the  lipase  content  of  liver  tissue  much  reduced  in  chloroform  necrosis 
(Jour.  Med.  Res.,  1915  (32),  73).  A  review  of  work  published  on  blood  changes 
and  liver  function  in  phosphorus  and  chloroform  poisoning  is  given  by  Marshall 
and  Rowntree,  Jour.  Exp.  Med.,  1915  (22),  333. 

"  Deut.  med.  Woch.,  1904  (30),  499. 

*' V.  Bergmann  (Hofmeister's  Beit.,  1904  (6),  40)  was  able  to  isolate  2.3  grams 
of  amino-acids  combined  with  tlie  chloride  of  naphthalene  sulphonic  acid,  from 
270  c.c.  of  blood  in  a  case  of  acute  vellow  atrophv. 

"  See  Van  Slyke,  Arch.  Int.  Med.,  1917  (19)," 77. 

»»  Biochem.  Zeit.,  1917  (79),  162. 


ACID  INTOXICATION  555 

200  mg.  per  100  c.c,  or  over  1  per  cent,  of  amino  acids.  The  urea  con- 
tent is  usually  below  50  mg.  The  lipin  content  of  the  blood  is  also 
greatly  increased, ^^  with  cholesteroleniia  but  a  decrease  in  phospho- 
lipins,  the  total  ether  extract  being  as  high  as  1.9  per  cent.  Sugar  is 
at  first  increased  and  later  decreased;  acetone  bodies  are  but  slightly 
increased. 

Originof  the  Ami  no=acids.—The  earliest  conception  of  the  source 
of  the  leucine  and  tyrosine  found  in  the  urine  was  that  it  came  from 
the  products  of  tryptic  digestion  a})sorbcd  from  the  intestinal  tract, 
which  the  liver  could  not  convert  into  urea  because  of  its  damaged 
condition.  On  the  demonstration  by  Jacoby^^  that  these  same  bodies 
were  present  in  the  livers  of  phosphorus-poisoned  animals  because  of 
autolysis,  it  became  probable  that  the  leucine  and  tyrosine  found  in 
the  urine  were  formed  from  the  degenerated  liver-cells  rather  than  in 
the  intestine,  which  view  has  become  generally  accepted.  It  seems 
most  probable,  however,  that  the  urinary  amino-acids  are  derived 
partly  (and  perhaps  chiefly)  from  the  autolysis  of  the  liver,  and 
partly  from  amino-acids  produced  both  in  the  intestine  and  within 
the  body  during  tissue  metabolism,  and  which  the  liver  cannot  trans- 
form into  urea  as  it  normally  does,  for  several  observers  have  reported 
that  even  relatively  slight  disturbances  in  hepatic  function  are  accom- 
panied by  a  considerable  rise  in  the  amino-acids  in  the  urine. ^^ 

ACID  INTOXICATION  AND  ACETONURIA" 
If  a  rabbit  is  given  in  repeated  small  doses  by  mouth  considerable 
quantities  of  inorganic  acids,  such  as  hydrochloric  or  phosphoric  acids, 
which  it  cannot  destroy  by  oxidation,  it  soon  becomes  extremely  ill. 
The  manifestations  are  characteristic — unsteadiness  of  motion  and 
stupor  being  followed  by  coma,  in  which  the  striking  feature  is  the 
excessively  active  respiration,  as  if  the  animal  were  being  asphyxiated 
(the  so-called  "air  hunger"),  while  at  the  same  time  there  is  no  cyanosis 
and  the  blood  is  bright  red,  containing  much  less  CO2  than  normal, 
while  the  amount  of  oxygen  remains  quite  normal.  The  current 
explanation  of  this  interesting  condition  is  as  follows:  Normally  the 
blood  carries  the  CO2  away  from  the  tissues  to  the  lungs  in  combina- 
tion with  the  inorganic  alkalies  of  the  blood,  of  which  sodium  is  by  far 
the  most  abundant.  This  combination  is  the  bicarbonate  of  sodium 
(or  other  base),  which  in  the  lungs  is  decomposed  into  the  carbonate, 
the  CO2  escaping  into  the  alveolar  air,  according  to  this  equation: 
2NaHC03  ?=i  NaoCOs  +  H2O  -|-  CO2 

"  IMd.,  1918  (86),  1. 

52  Zeit.  phvsiol.  Chem.,  1900  (30),  174. 

"  See  Masuda,  Zeit.  exp.  Path.,  1911  (8),  629;  Labb6  and  Bith,  Compt.  Rend. 
Soc.  Biol.,  1912  (73),  210. 

"  General  lituraturp  to  1908,  given  by  Ewing,  Arch.  Int.  Med.,  1908  (2),  330; 
also  see  Magnus-Levy,  Ergebnisse  inn.  Med.,  1908  (1),  374;  Lusk,  Arch.  Int. 
Med.,  1909  (3),  1.  More  recent  literature  given  by  Hurtley,  (Juart.  Jour.  Med., 
1916  (9),  301,  and  see  also  monograph  by  Sellards,  "Principles  of  Acidosis,' 
Harvard   Univ.  Press,  1917;  Whitnev,  Bost.  Med.  Surg.  Jour.,  1917  (176),  225. 


556  ABNORMALITIES  IN  METABOLISM 

The  carbonate  thus  formed  goes  back  to  the  tissues  to  combine  again 
with  more  CO2  and  form  bicarbonate.  If  acids  are  introduced  into 
the  blood  they  combine  with  the  alkahes  there,  forming  neutral  salts 
which  are  ehminated  in  the  urine,  and  in  this  way  the  amount  of 
alkali  in  the  blood  is  reduced,  with  a  consequent  reduction  in  the 
capacity  of  the  blood  to  carry  CO2  away  from  the  tissues;  the  amount 
of  CO2  in  the  blood  sinking  to  as  low  as  2.5  and  3  per  cent.  (Walter). 
Consequently,  in  acid  poisoning  the  CO2  produced  in  metabolism  ac- 
cumulates in  the  tissues  where  it  is  formed,  and  blocks  the  processes 
of  oxidation,  so  that  the  animal  suifers  from  asphyxia  exactly  as  if  it 
were  deprived  of  air.  In  other  words,  the  lack  of  alkalies  in  the  blood 
in  acid  intoxication  checks  the  "internal  respiration,"  as  intracellular 
gas  exchange  is  called,  by  preventing  the  removal  of  CO2  from  the  cells. 
The  acids  stimulate  the  respiratory  center,  which  is  extremely  sensitive 
to  them,  and  the  increased  respiration  tends  to  reduce  the  aciditA^  by 
getting  rid  of  the  CO2,  but  under  the  conditions  of  the  experiment 
this  is  not  sufficient  to  prevent  asphyxia. 

If  the  urine  of  such  an  animal  is  analyzed,  it  is  found  to  contain 
increased  quantities  of  the  four  chief  inorganic  bases,  Na,  K,  Ca,  and 
Mg  (the  last  two  apparently  being  derived  from  the  bones) ;"  but  in 
addition  to  these  it  is  found  that  the  amount  of  ammonia  in  the  urine 
is  decidedly  increased.  If  instead  of  a  rabbit  a  carnivorous  animal, 
such  as  a  dog,  is  given  acids,  it  will  be  found  relatively  insusceptible, 
so  that  much  larger  quantities  can  be  given  without  causing  acid 
intoxication.  Examination  of  the  urine  of  such  a  dog  will  show  that 
the  elimination  of  ammonia  is  increased  much  more  than  it  is  in  the 
herbivora,  while  the  inorganic  alkalies  are  increased  but  little.  From 
this  it  is  deduced  that  in  acid  intoxication  part  of  the  nitrogen  that 
normally  goes  to  form  urea  becomes,  while  in  the  antecedent  form  of 
ammonia,  combined  with  part  of  the  acid  that  has  entered  the  blood. 
In  this  way  much  of  the  neutralization  of  the  acids  is  accomplished 
by  ammonia,  and  the  inorganic  alkalies  of  the  blood  are  spared.  As 
in  carnivora  the  amount  of  protein  metabolism  is  much  greater  and 
more  rapid  than  in  herbivora,  the  ammonia  available  for  neutral- 
ization of  acids  is  much  greater  than  in  the  latter,  and  hence  the  rela- 
tive lack  of  susceptibility  of  carnivora  to  acid  poisoning.'^''  The 
proteins  of  the  blood  also  combine  some  of  the  acid,  perhaps  one-fifth 
of  the  neutralizing  capacity  of  the  ])lood  being  attributable  to  tliom. 
Another  factor  is  the  possible  accunuilation  of  acids  within  the  cells, 
which  must  modify  greatly  any  conclusions  based  upon  studies  of  the 
blood  and  urine.  It  is  within  the  cells  that  the  effects  of  acids  must 
be  manifested,  and  it  is  perfectly  possible,  and  indeed  almost  certain, 

"  See  Goto,  Jour.  Biol.  Clieni.,  1918  (36),  355. 

'*  This  hiis  been  nicely  shown  by  Eppingor  (^^'i('n.  kliii.  \\'oi'li.,  ll>()('»  (H>),  111), 
who  found  that  udministnition  of  considerahle  quantities  of  amino-acids  (glycine 
alanine,  aspartic  acid)  to  ral)])its  greatly  increased  tiieir  resistance  to  acid  intoxica- 
tion, i)resunial)]y  by  yielding  ninnionia  through  normal  stc'jis  of  protein  metabolism. 


ACIDOSIS  557 

that  we  may  have  degrees  of  acidity  and  alkalinity  in  the  cells  which 
are  quite  different  from  those  in  the  blood. 

As  pointed  out  especially  by  Henilerson,"  the  normal  reaction  of 
the  body  is  kept  practically  constant  chiefly  by:  1.  The  salts  of  (.'O2 
and  H3PO4,  existing  in  such  proportions  of  carbonate,  bicarbonate  and 
carbonic  acid,  or  disodium-  and  monosodium-hydrogen-phosphate, 
as  to  produce  an  almost  neutral  solution.  These  being  salts  of  weak 
acids  with  strong  bases  it  follows  that  when  a  stronger  acid,  such  as 
lactic  or  butyric,  combines  with  the  bases  there  is  only  the  weak  acid 
liberated  and  hence  the  influence  of  the  strong  acid  on  the  blood  reac- 
tion is  greatly  reduced.  (2)  The  acid  most  abundantly  formed  in 
metabolism,  COo,  is  volatile  and  hence  is  rapidly  excreted  by  the  lungs 
without  withdrawing  bases  from  the  blood.  (3)  The  kidneys  can 
eliminate  the  other  buffer  acid,  PO4,  with  l)ut  a  minimum  of  base  at- 
tached in  the  form  of  NaH2P04;  and  they  also  remove  the'  basic 
product  of  metabolism,  ammonia.  By  the  combined  influence  of  these 
factors  the  acids  formed  in  metabolism  are  passed  out  with  a  maxi- 
mum rapidity  and  with  a  minimum  alteration  in  the  reaction  of  the 
fluids  by  which  they  are  carried  through  the  body.  In  addition  to 
these  we  have,  as  mentioned  before,  the  capacitj^  of  the  proteins  to 
combine  with  both  acids  and  alkalies,  the  reserve  neutrahzing  capacity 
of  ammonia  formed  in  metabolism,  and  also  the  enormous  reserve  supply 
of  bases  in  the  bone  salts.  So  effective  is  this  mechanism  that  accu- 
rate determination  of  the  H-ion  concentration  of  the  blood  shows  that 
very  rarely  is  there  more  than  the  slightest  deviation  from  the  normal 
proportion  of  free  H  and  OH  ions,  which  is  slightly  on  the  alkaline  side 
of  exact  neutrality.  This  neutrality  is  one  of  the  most  fixed  of  all  the 
constants  of  the  body. 

Acidosis,  therefore,  is  a  condition  in  which  the  essential  feature  is 
not  an  actual  acidity  of  the  blood,  but  the  impoverishment  of  the  body 
in  available  bases,  whereby  there  results  a  decreased  capacity  of  the 
tissues  to  get  rid  of  CO2  and  other  acids  formed  in  their  metabolism. 
This  reduction  in  bases  may  be,  and  most  usually  is,  the  result  of 
excessive  production  of  acids,  in  excreting  which  the  bases  are  elimi- 
nated in  excess,  but  it  may  also  result  from  deficient  capacity  of  the 
kidneys  to  excrete  acids,  since  the  kidneys  plaj'  an  important  role  in 
regulating  acidity.  Macleod^^  summarizes  the  conditions  that  might 
give  rise  to  changes  in  the  hj'drogen  ion  concentration  in  the  blood 
(Ch)  as  follows : 

Increase  of  Ch. 
Addition  or  accumulation     Accumulation    of    CO2    (asphyxial     conditions).     In- 
0/  acid  complete  oxidation   of  carbohydrate    (lactic  acid  in 

muscular  exercise). 
Defective  oxidation  of  fat  (ketosis). 
Renal  insufficiency  (nephritis). 
Decomposition  of  protein  (as  in  acidosis  of  fever). 
Intestinal  fermentation. 
Administration  of  acid  (experimental). 

*^  See  Harvey  Society  Lectures,  1914-5. 

"  See  reviews  by  Frothingham,  Arch.  Int.  Med.  1916  (18)  717;  ^klacleod,  Jour. 
Lab.  Clin.  Med.,  1919  (4),  315. 


558  ABNORMALITIES  IN  METABOLISM 

Increase  of  Ch. 

Decrease  of  base  Diarrhea  and  hemorrhage,   respectively  (may  explain 

acidosis  in  cholera  and  in  certain  forms  of  shock).*' 

Decrease  in  Ch. 
Addition  or  accumulation     Ammonia  (faulty  metabolism  of  urea). 

of  base  Intestinal  putrefaction  (infantile  conditions). 

Administration  of  alkalies  (experimental). 
Removal  of  adds  Excretion  of  CO2  (excessive  pulmonary  ventilation,  as 

in  faulty  ether  administration). 
Excretion  of  acid  urine. 

Practically,  acidosis  results  either  from  defective  oxidation  of 
organic  acids  formed  in  metabolism  or  from  defective  elimination  of 
mineral  acids  (acid  phosphate)  because  of  impaired  renal  function. 
The  chief  example  of  the  former  is  the  acidosis  of  diabetes,  of  the 
latter  the  acidosis  of  nephritis,  and  mixed  forms  may  occur. 

The  degree  of  acidosis  may  be  estimated  in  several  ways;  as 
follows  :^^ 

1.  By  determining  the  CO2  content  of  the  blood,  which  must  decrease  as  other 
acids  increase,  or  as  the  bases  decrease. 

2.  Direct  estimation  of  the  H-ion  concentration  of  the  blood. 

3.  By  determining  the  amount  of  acid  or  alkali  necessary  to  change  the  reaction 
of  the  blood  to  different  indicators. 

4.  Determination  of  the  CO2  tension  of  the  alveolar  air,  this  varying  directly 
with  the  CO2  tension  of  the  arterial  blood. 

5.  The  "alkali  tolerance  test"  of  Sellards,  which  consists  in  ascertaining  the 
amount  of  sodium  bicarbonate  that  must  be  taken  by  mouth  in  order  to  produce 
an  alkaline  urine. 

6.  Estimation  of  the  amount  of  organic  acids,  H-ion  concentration,  and 
ammonia  content  of  the  urine;  a  method  which  is  fundamentallj^  defective  since 
it  indicates  merely  the  acids  and  bases  that  have  been  removed  from  the  body 
and  not  those  that  remain  to  modify  its  reactivity. 

7.  Determination  of  the  capacity  of  the  blood  serum  to  bind  CO2.  Normal 
serum  binds  about  55  to  75  per  cent,  of  its  volume  of  CO2,  whereas  in  acidosis  it 
may  bind  but  20  per  cent.  (Van  Slyke) 

Diabetic  Coma^* 

In  man,  poisoning  with  inorganic  acids,  as  in  the  experiments  cited 
above,  is  a  rare  occurrence,  but  not  infrequently  acid  intoxication  re- 
sults from  the  presence  of  undue  quantities  of  organic  acids  produced 
in  metabolism.  The  most  striking  example  of  this  is  the  coma  of 
diabetes,  in  which  the  asphyxia  without  cyanosis,  dependent  upon  fail- 
ure of  the  blood  to  carry  CO2,  is  sometimes  strikingly  similar  to  that 
observed  in  experimental  animals  poisoned  with  acids.  In  diabetic 
coma  the  acid  intoxication  is  due  chiefly  to  the  accumulation  in  the 
blood  or  tissues  of  large  quantities  of  ^-oxybutyric  acid.  Associated 
with  it,  in  smaller  quantities,  are  usually  found  diacetic  (accioocctic) 
acid  and  acetone,  which  are  chemically  so  closely  related  that  it  has  been 

^^  To  thi.s  may  be  added  loss  of  base  from  biliary  or  pancreatic  fistulse. 


DIABETIC  COMA  559 

generally  considered  that  they  are  derived  from  the  oxybutyria  acid, 
as  follows: 

/3-oxybutyric  acid  is — 

CH^CHOH— CH2— COOH, 

and  by  oxidation  this  readily  forms — 

CH3— CO— CHa— COOH, 

which  is  diacetic  acid  (being  two  molecules  of  acetic  acid  united  to 
each  other,  as  follows) : 


CH3— CO— |0H— Hj— H2C— COOH. 

Diacetic  acid  is,  in  turn,  readily  deprived  of  its  carbon  dioxide,  forming 
acetone, 

CH3— CO— CH3. 

All  these  reactions  are  easily  accomplished  in  the  laboratory,  and  there 
seemed  to  be  reason  for  believing  that  they  may  normally  occur  in  the 
same  way  in  the  animal  body.  Wakeman  and  Dakin,^^"  and  others, 
however,  found  evidence  that  the  liver  cells  may  also  reduce  diacetic 
to  i3-oxybutyric  acid,  and  it  seems  probable  that  this  is  the  usual 
direction  of  the  reaction,  which  they  have  found  to  be  produced  by  a 
specific  enzyme.  Hurtley^^  concludes  that  the  reduction  of  aceto- 
acetic  acid  to  oxybutyric  acid  is  accomplished  by  the  body  under 
ordinary  conditions  far  more  readily  than  the  oxidation  of  oxj^butyric 
to  aceto-acetic  acid.  Marriot*^"  gives  the  following  scheme  as  indicat- 
ing the  normal  path  of  fatty  acid  catabolism : 

d-oxj'butyric  acid 
Fatty  acid >Butyric  acid(?) > Aceto-acetic  acid '\l-oxybutyri™acid 

(difficultly  burned) 

The  study  of  the  utilization  of  the  acidosis  substances  when  injected 
intravenously  at  accurately  measured  rates  for  considerable  periods 
by  Wilder,  ^^  furnishes  conclusive  evidence  of  the  origin  of  /3-oxy- 
butyric acid  from  acetoacetic  acid.  He  found  that  normal  dogs  ex- 
crete i3-oxybutyric  acid  when  it  is  injected  at  the  rate  of  0.4  gm. 
(0.0032  gm.  molecule)  of  the  sodium  salt  per  kilo  of  body  weight  per 
hour,  but  not  with  lower  rates.  Sodium  acetoacetate  was  excreted 
when  injected  in  rates  of  0.2  gm.  (0.0016  gm.  molecule)  per  kilo  per 
hour,  and  when  injected  at  the  0.4  gm.  rate  it  was  excreted  accom- 
panied by  /3-oxybutyric  acid.  Evidently,  then,  the  acetoacetic  acid 
must  be  converted  almost  quantitatively  into  /3-oxybutyric  acid.  On 
the  other  hand,  acetoacetic  acid  did  not  appear  in  the  urine  when 
larger  quantities  of  /3-oxybutryic  acid  were  injected,  and  hence  it  is 

59-  Jour.  Biol.  Chem.,  1909  (6),  373;  1910  (8),  105. 

60  Jour.  Biol.  Chem.,  1914  (18),  241. 

61  Jour.  Biol.  Chem.,  1917  (31),  59. 


560  ABNORMALITIES  IN  METABOLISM 

apparent  that  not  much  if  any  urinary  acetoacetic  acid  is  derived  from 
this  source. 

As  long  as  a  normal  individual  is  burning  at  least  one  molecule  of 
carbohydrate  to  three  of  higher  fatty  acids  the  urine  is  free  from  more 
than  traces  of  these  three  "acetone  bodies,  "'^-  but  when  for  any  reason 
daily  oxidation  of  carbohydrates  falls  below  this  minimum  the  two  the 
acids  appear,  combined  largely  with  ammonia,  but  partly  with  mineral 
bases.  Fats  burn  in  the  fire  of  the  carbohydrates,  and,  as  Wood- 
yatt*^^  puts  it,  when  the  proportion  of  fat  is  too  great  for  the  fire 
it  "smokes"  with  unburnt  fats  and  acetone  bodies.  Normally  but  2 
to  5  per  cent,  of  the  nitrogen  of  the  urine  is  in  the  form  of  ammonia, 
but  in  diabetic  acidosis  the  proportion  may  reach  from  10  to  25  per 
cent.,  the  proportion  of  urea  being  correspondingly  reduced.^'* 

The  presence  of  large  quantities  of  these  acids  in  the  urine  presages 
coma,  during  which  the  amount  of  oxybutyric  acid  often  reaches  15-20 
grams  per  day,  and  has  been  known  to  reach  150  grams  (Kiilz  claimed 
to  have  found  226  grams).  Diacetic  acid  appears  in  relatively  small 
amounts,  rarely  exceeding  10  per  cent,  of  the  total  organic  acids  of 
the  urine ;^^  as  a  rule,  when  any  one  of  the  three  acetone  bodies  is 
present  in  large  amounts,  there  is  an  abundance  of  each  of  the  others. 
Kenneway'''^  confirms  Neubauer's  statement  that  oxybutyric  acid  is 
rather  constantly  from  60  to  80  per  cent,  of  the  total  acetone  bodies 
excreted  in  the  urine.  In  the  internal  organs  the  acetone  bodies  may 
also  be  detected,  especially  in  the  liver."  In  normal  blood  Marriott 
found  less  than  4  mg,  of  oxybutyric  acid,  and  1.5  mg.  of  acetone  and 
aceto-acetic  acid  together,  per  100  c.c;  but  in  diabetic  coma  the 
figures  rose  as  high  as  45  mg.  and  28  mg.  respectively  for  each  fraction, 
the  amount  in  the  blood  not  corresponding  to  the  urinary  excretion,  ^^  or 
to  the  bicarbonate  content  of  the  blood. "^^  Associated  with  acidosis  is 
usually  an  increase  in  the  blood  lipins.'^" 

Relation  of  Acidosis  to  Diabetic  Coma. — There  seems  to  be 
httle  room  for  doubt  that  the  typical  diabetic  coma  with  "air  hunger" 

^^JVeeder  and  Johnson  (Amer.''Jour.  Dis.  Chil.,  1916  (11),  291)  give  as  the  nor- 
mal daily  average  excretion  32  mg.  of  ketones  (diacetic  acid  and  acetone)  and  38 
mg.  oxybutyric  acid.  The  old  statement  that  acetone  appears  in  advance  of  tlie 
two  acids  is  incorrect,  the  error  being  due  to  faulty  methods  (See  Howhind  and 
Marriott,  Amer.  Jour.  Dis.  Chil.,  1916  (12),  459).  Concerning  normal  occurrence 
of  acetone  in  blood  and  tissues,  see  Halpern  and  Landau,  Zeit.  exp.  Path.  u.  Ther., 
1906  (3),  466. 

63  Jour.  Amer.  Med.  Assoc,  1916  (66),  1910. 

"According  to  Edie  and  Whitley  (Biochemical  Jour.,  1906  (1),  11),  adminis- 
tration of  excessive  amounts  of  alkali  causes,  conversely,  elimination  of  increased 
amounts  of  organic  acids. 

6^  Folin  says  that  perfectly  fresh  diabetic  urine  does  not  contain  any  acetone, 
that  which  is  commonly  found  being  derived  from  diacetic  acid  which  rapidly 
decomposes  into  acetone. 

""  Biochem.  Jour.,  1913  (8),  355. 

"  Sassa,  Biochem.  Zeit.,  1914  (59),  362. 

««  Marriott,  Jour.  Biol.  Chcm.,  1914  (18),  507. 

^Titz,  Trans.  Assoc.  Amer.  Phys.,  1917  (32),  155. 

'">  Gray,  Boat.  Med.  Surg.  Jour.,  1917  (178),  16-156. 


DIABETIC  COMA  561 

depends  upon  an  excess  of  these  substances  in  the  blood — i.  e.,  is 
an  acid  intoxication — for  the  followinj^  reasons:  (1)  The  coma  usually 
appears  when  the  amount  of  organic  acids  in  the  urine  is  highest,  and 
is  absent  when  there  is  little  or  none  of  them  in  the  urine.  (2)  Be- 
cause of  the  resemblance  of  the  symptoms  to  those  of  experimental  acid 
intoxication.  (3)  Because  of  the  repeated  demonstration  of  a  re- 
duced amount  of  alkali  in  the  blood,  as  determined  by  titration,  and 
a  great  reduction  of  the  amount  of  CO2  carried  in  the  venous  blood. 
The  capacity  of  the  serum  to  absorb  CO2  in  vitro  is  also  greatly  re- 
duced, from  a  normal  55  to  75  per  cent,  to  as  low  as  20  per  cent.  (Van 
Slyke).  By  means  of  gas-chain  measurements  Roily ^'  found  that 
by  far  the  lowest  OH  values  ever  observed  in  the  blood  are  in  dia- 
betic coma;^-  and  Sellards^^  showed  that  in  diabetes  the  tolerance 
for  alkalies  may  be  increased.  He  states^'*  that  a  deficit  of  20  to  30 
grams  of  sodium  bicarbonate  produces  a  degree  of  acidosis  demonstra- 
ble by  blood  examination  onlj'-,  40  to  50  grams  deficit  usually  ciiuses 
only  dyspnoea  on  exertion,  75  to  100  grams  deficit  may  produce 
persistent  dyspnoea,  150  grams  deficit  may  accompany  coma,  while 
the  maximum  deficits  reported  have  been  about  200  grams.  (4) 
The  marked  improvement  that  sometimes  results  from  the  administra- 
tion of  alkalies  (usually  sodium  bicarbonate).  Associated  with  this 
improvement  is  an  elimination  of  greatly  increased  amounts  of  organic 
acids,  indicating  their  previous  retention  in  the  body  because  of  lack 
of  alkali  with  which  they  could  combine. 

But  there  are  cases  of  diabetic  coma  without  typical  air  hunger, 
and  it  is  the  exception  rather  than  the  rule  for  alkali  therapy  to 
produce  a  marked  improvement  in  the  fully  developed  coma  of 
diabetes.  Furthermore,  coma  may  occur  in  diabetics  who  are  pro- 
ducing no  such  quantity  of  organic  acids  as  would  seem  theoreti- 
cally to  be  necessary  to  cause  enough  acid  intoxication  to  result  in 
acidosis,  and  coma  develops  in  diabetics  who  are  being  supphed  with 
sufficient  bases  for  all  requirements.  Hence  it  must  be  concluded 
that  only  a  part  of  the  symptomatology  of  diabetic  coma  depends  on 
acids  as  such,  but  as  yet  we  do  not  know  what  other  agents  are  acting.^'* 

/3-oxybutyric  and  diacetic  acid,  according  to  many  authorities, 
seem  to  have  no  specific  poisonous  effects,  but  act  simply  as  acids 
in  the  blood.  Acetone  does  not  have  this  eff'ect,  not  being  an  acid, 
and  seems  not  to  be  toxic  to  any  considerable  degree;  doses  of  4  grams 
per  kilo  cause  effects  similar  to  ethyl  alcohol  in  dogs,  8  grams  per 
kilo  being  fatal,  which  corresponds  to  a  dose  of  500  grams  for  an  adult 

'1  Munch,  med.  Woch.,  1912  (59),  1201. 

^^  Menten,  however,  reports  that  in  diabetes  before  acidosis  the  OH  concen- 
tration is  usually  somewhat  above  normal  (Jour.  Cancer  Res.,  1917  (2),  179). 

^3  Johns  Hopkins  Hosp.  Bull.,  1912  (23),  289. 

^■*  Pribram  and  Loewy  (Zeit.  klin.  Med.,  1913  (77),  384)  suggest  that  abnor- 
mal products  of  protein  cleavage  are  responsible,  and  Rosenbloom  (N.  Y.    Med. 
Jour.,  Aug.  7,  1915)  reports  cases  of  typical  diabetic  coma  without  acetone  bodies 
in  the  urine. 
36 


562  ABNORMALITIES  IN  METABOLISM 

man.  According  to  Rhamy^*  acetone  is  more  toxic  (for  guinea-pigs) 
than  methyl  alcohol,  while  for  rabbits  Desgrez  and  Saggio^^  found 
acetone  the  least  toxic  of  the  acetone  bodies,  diacetic  acid  next,  and 
j8-oxybutyric  acid  most.  Ehrmann'^  also  claims  that  he  has  pro- 
duced typical  coma  with  the  sodium  salts  of  butyric  and  of  /3-oxybuty- 
ric  acid,  but  as  high  as  40  grams  of  /3-oxybutyric  acid  have  been  found 
in  the  day's  urine  of  a  non-diabetic  without  any  evidence  of  intoxica- 
tion. Ewing  suggests  that  the  acetone  bodies  may  cause  renal 
injury,  which  is  usually  evident  in  acidosis,  and  M.  H.  Fischer's 
views  on  the  relation  of  acids  to  nephritis  accord  with  this  fact.  The 
withdrawal  of  the  inorganic  bases,  especially  Ca  and  Mg,  may  also 
be  responsible  for  symptoms,  as  it  is  well  established  that  a  proper 
balancing  of  these  ions  is  necessary  for  normal  cell  activity,  especially 
in  the  nervous  system.''^ 

Hurtley^^  sums  up  the  evidence  on  the  toxicity  of  the  acetone 
bodies  by  saying  that  aceto-acetic  acid  seems  to  be  highly  toxic  only 
in  depancreatized  animals,  while  oxybutyric  acid  is  practically  non- 
toxic. He  favors  the  view  that  aceto-acetic  acid  poisoning  is  respon- 
sible for  diabetic  coma,  for  it  increases  in  the  urine  on  the  approach 
of  coma,  and  the  ratio  of  aceto-acetic  to  butyric  acid  in  the  urine  in- 
creases with  the  severity  of  the  intoxication.  The  increased  propor- 
tion of  aceto-acetic  acid  presumably  means  that  it  is  being  produced 
in  such  quantities  throughout  the  body  that  the  liver  cannot  reduce 
as  large  a  proportion  to  oxybutyric  acid  as  it  normally  does.  In  con- 
sidering the  possibility  that  the  acetone  bodies  may  be  responsible  for 
at  least  part  of  the  intoxication  of  diabetic  coma  we  must  bear  in 
mind  that  the  evidence  of  their  low  toxicity  is  based  on  short  time 
experimental  intoxications,  and  that  they  may  be  found  to  be  much 
more  toxic  than  is  generally  assumed  when  they  are  allowed  to  act 
for  many  days  and  weeks  on  the  nervous  tissues,  as  thej'  do  in  dia- 
betes. That  is,  the  experimental  evidence  concerning  the  toxicitj'  of 
the  acetone  bodies  has  not  been  obtained  under  conditions  comparable 
to  those  of  diabetic  acidosis. 

Origin  of  the  Acetone  Bodies. — The  chemical  nature  of  the  ace- 
tone bodies  is  such  that  they  might  readily  be  produced  from  any  or 
all  of  the  three  classes  of  foodstuffs. 

They  might  be  derived  from  carbohydrates,  as  is  the  closely  related  lactic  acid, 
but  we  know  that  this  it  not  the  usual  source.  On  the  contrary,  administration  of 
a  proper  amount  of  carbohydrates  under  certain  conditions  may  cause  tlie  acids 
to  disappear  from  the  urine,  and  acetone  bodies  may  be  cHminated  in  large  quan- 
tities while  the  patient  is  on  a  diet  ahnost  free  from  carbohydrates.  Carbohy- 
drates are,  indeed,  the  most  active  agents  in  preventing  the  formation  of  these 
ketone  bodies,  i.  e.,  they  are  antiketogenic.''* 

"  Jour.  Amer.  Med.  Assoc,  1912  (58),  628. 
'8  Compt.  Rend.  Soc.  Biol.,  1907  (63),  288. 
"  Berl.  klin.  Woch.,  1913  (50),  11. 
"See  Cammidge,  Amer.  Med.,  1916  (11),  363. 

"  Concerning  antiketogencsis  see  Woodyatt,  Jour.  Amer.  Med.  Assoc,  1910 
55),  2109. 


ACIDOSIS  AND  ACETONURIA  563 

They  might  readily  be  formed  from  proiciiiN  through  splitting  out  of  the  NHj 
group  from  the  amino-acids;  indeed  the  amino-acids  arc  generally  considered  as  a 
source  of  the  acetone  bodies/"  particularly  because,  whenever  there  is  considerable 
pathological  breaking-down  of  proteins,  these  bodies,  especially  acetone,  may 
appear  in  the  urine;  c.  g.,  during  absorption  of  exudates,  in  carcinoma,  and  in 
starvation  or  other  conditions  with  great  wasting  of  the  tissues.  Dakin"  has 
shown  that  only  leucine,  histidine,  plienylalaninc  and  tyrosine  yield  diacetic  acid 
when  perfused  through  the  liver,  while  most  of  the  other  amino-acids  are  able  to 
yield  sugar  in  diabetic  animals,  and  hence  are  antiketogenic. 

On  the  other  hand,  the  amount  of  acids  sometimes  found  in  the  urine  seems  to 
be  greater  than  can  be  explained  by  the  protein  destruction  that  occurs  (Magnus- 
Lev}0>*^  and  in  diabetes  it  is  often  observed  that  feeding  of  fats  and  fatty  acids 
increases  the  output  of  acetone  bodies,  and  hence  it  is  evident  that  acetone  bodies 
may  be  derived  from  the  fats.  /3-oxybiitj'ric  acid  can  be  produced  readily  from 
fatty  acids,  especially,  of  course,  from  butyric  acid,  and  we  usually  observe  an  in- 
crease in  the  acetone  excretion  in  a  diabetic  given  large  quantities  of  butter.  Other 
higher  fatty  acids  are  also  found  to  cause  increased  acetone  excretion. 

The  studies  of  Knoop,^'  and  his  associates  have  indicated  that  in  the  cata- 
bolism  of  fatty  acids,  the  chains  are  broken  down  by  oxidation  of  the  carbon  atom 
third  from  the  end,  that  is,  the  /3-position,  and  the  two  end  carbon  atoms  are  then 
split  off.  Therefore,  two  carbon  atoms  are  always  split  off  at  a  time,  and  hence 
there  can  be  oxidized  into  oxybutyric  acid  only  those  fatty  acids  which  contain 
an  even  number  of  carbon  atoms,  which  includes  the  ordinary  fatty  acids  (oleic, 
palmitic  and  stearic)  of  fat  tissue,  which  have  each  an  even  number  of  carbon 
atoms  (16  or  18),  and  also  butyric,  caproic  and  similar  acids.  Normal  fatty  acids 
which  contain  an  odd  number  of  carbon  atoms  cannot  yield  oxybutyric  acid. 
However,  according  to  A.  Loeb,*^  aceto-acetic  acid  may  be  built  up  from  acetic 
acid  in  the  liver,  and  the  urine  in  diabetes  may  contain  acetic  acid.  "The  forma- 
tion of  oxj-butyric  acid  and  of  diacetic  acid  in  all  these  cases  may  be  said  to  be  due 
to  the  fact  that  the  diabetic  organism  is  not  able  quite  to  finish  the  attack  on  the 
beta-carbon  atom  of  butj-ric  acid"  (Folin). 

From  the  results  of  these  studies  it  seems  that  the  acetone  bodies  can,  theoreti- 
cally be  formed  from  any  of  the  three  classes  of  food-stuffs,  but  that  ordinarily 
they  come  chiefly  from  the  fats,  and  in  severe  diabetes  also  to  a  considerable  extent 
from  fatty  acids  formed  by  deaminization  of  amino-acids.  Although  it  is  prob- 
able that  the  acetone  bodies  are  formed  in  many  if  not  all  tissues,  yet  there  is 
abundant  evidence  that  the  liver  plays  an  important  part  in  ketogenesis,  as  shown 
by  the  decrease  in  acetone  bodies  in  Eck  fistula  dogs,  and  their  great  increase  when 
the  blood  supply  of  the  liver  is  augmented.** 

ACIDOSIS    AND  ACETONURIA  IN   CONDITIONS   OTHER  THAN 

DIABETES"" 

When  our  chief  method  of  recognition  of  acidosis  consisted  of 
determining  the  presence  of  acetone  bodies  in  the  urine,  the  term 
acetonuria  was  used  as  synonymous  with  acidosis,  but  we  now  know 
that  we  may  have  varying  degrees  of  acetonuria  without  significant 

*"  Embden  and  his  associates  have  (Hofmeister's  Beitr.,  1906  (8),  121;  1908 
(11),  H.  7-9)  demonstrated  that  the  liver  can  form  acetone  from  many  substances 
perfused  through  it  in  the  blood,  including  not  only  amino-acids  of  the  fatty 
acid  series,  but  also  the  aromatic  radicals  of  the  protein  molecule. 

81  Jour.  Biol.  Chem.,  1913  (14),  328. 

82  Arch.  exp.  Path.  u.  Pharm.,  1899  (42),  149;  Ergeb.  inn.  Med.,  1908  (1), 
374. 

83  Full  bibliography  and  discussion  by  Porges,  Ergebnisse  Physiol.,  1910  (10), 
6.  See  also  Ringer,  Jour.  Biol.  Chem.,  1913,  Vol.  14. 

8<  Biochem.  Zeit.,  1912  (47),  118. 

85 Fischer  and  Kossow.  Deut.  Arch.  klin.  Med.,  1913  (101),  479. 
85<»  See  Sellards  "Principles  of  Acidosis,  Harvard  Press,  1917;  also Frothingham, 
Arch.  Int.  Med.,  1916  (18),  717. 


564  ABNORMALITIES  IN  METABOLISM 

acidosis  as  determined  by  examination  of  the  blood,  and  severe  acidosis 
may  occur  with  Uttle  or  no  acetonuria. 

Furthermore,  we  may  have  high  urinary  ammonia  not  only  be- 
cause of  excretion  of  diacetic  and  oxybutyric  acids  as  in  diabetic 
acidosis,  but  also  from  excretion  of  excessive  quantities  of  lactic  acid, 
as  in  puerperal  eclampsia,  pernicious  vomiting  of  pregnancy,  acute 
yellow  atrophy,  and  other  conditions  associated  with  severe  injury  to 
the  liver.  As  high  ammonia  excretion  is  characteristic  of  diabetic 
acidosis,  the  presence  either  of  acetone  bodies  or  high  urinary 
ammonia  was  formerly  considered  to  indicate  the  existence  of  acidosis, 
but  it  is  now  recognized  that  as  a  general  rule  there  is  not  a  severe 
acidosis  in  these  hepatic  diseases,  the  intoxication  being  dependent 
on  neither  lactic  acid  nor  acidosis,  but  upon  poisons  of  unknown 
character.  This  group  of  diseases  has  been  considered  in  previous 
pages.  Because  of  its  significance  as  one  of  the  chief  organic  acids 
formed  in  metabolism,  rather  than  as  a  cause  of  serious  acidosis,  we 
may  in  this  chapter  briefly  consider  lactic  acid  and  its  relation  to 
disease. 

Sarcolactic  Acid  often  is  found  in  the  urine,  but  in  origin  and  significance  it  is 
entirely  different  from  the  acetone  bodies,  and  it  probably  is  never  present  in 
sufficient  amounts  to  cause  an  acid  intoxication  by  abstraction  of  alkalies  from  the 
blood.  In  vitro,  we  obtain  sarcolactic  acid  whenever  sugar  is  placed  in  an  alkaline 
solution,  provided  the  siipply  of  oxygen  to  the  solution  is  deficient;  but  if  the  oxygen 
supply  is  adequate,  sugar  will  not  yield  lactic  acid  with  alkalies  (Nef).  Similarlj', 
an  isolated  surviving  muscle,  when  asphyxiated  by  any  means,  shows  a  rapid  ac- 
cumulation of  lactic  acid,  which  fails  to  occur  when  sufficient  oxygen  is  supplied. 
This  lactic  acid  comes  chiefly  from  sugar,  but  about  25  to  30  per  cent,  of  it  can  have 
its  origin  in  protein  (or  fat?)  (Woodyatt).  If  an  organism  as  a  whole  is  insuffi- 
ciently supplied  with  oxygen,  lactic  acid  accumulates  in  the  tissues  and  appears 
in  the  urine,  disappearing  when  the  oxygen  supply  is  restored.  Lactic  acid  often 
appears  after  poisoning  with  a  large  number  of  drugs,  which  Loewy  has  classified 
as  drugs  whose  action  in  the  body  resembles  that  of  lack  of  oxygen  (arsenic,  phos- 
phorus, hydrazine,  chloroform,  etc.).  These  poisons  are  all  characterized  by 
causing  impoverishment  of  glycogen,  fatty  liver,  and  acute  degenerative  changes 
especially  in  the  liver  cells  and  the  endothelium.  Therefore  the  assumption  seems 
justified  that  the  poisons  and  conditions  which  lead  to  lactic  acid  excretion  depend 
ultimately  upon  impairment  of  the  interchange  of  oxygen  in  the  cells.  Wood- 
yatt states  that,  as  far  as  known,  lactic  acid  has  never  been  demonstrated  in  any 
tissue  in  which  deficient  oxygenation  can  be  excluded,  and  regaids  lactic  acid  as 
the  metabolite  of  asphyxia  or  its  equivalent.  Over  against  this  view  is  that  of 
Embden  and  his  associates,  which  is  shared  by  others,  that  lactic  acid  is  a  normal 
intermediary  in  the  breakdown  of  the  sugars  in  the  bodj^  its  direct  anteceilent 
being  a  triose,  but  perusal  of  their  work  only  emphasizes  that  in  all  the  conditions 
in  which  their  data  were  obtained  asphyxial  conditions  were  present;  furtliennore, 
this  conception  of  lactic  acid  as  a  chief  intermediate  in  normal  sugar  cataluilism 
is  not  in  liarmony  with  the  best  ideas  of  carbohydrate  cliemistry  (\\'oodyatt). 
This  avitlior  lias  furthermore  found,  by  direct  observation  of  tlie  utilization  of 
lactic  acid  when  injected  intravenously,  that  it  cannot  well  bo  an  important  inter- 
mediate in  carbohydrate  catabolism.*"  (See  also  discussion  of  lactic  acid  under 
Diabetes,  Chapter  xxiv). 

It  is  possible  that  the  presence  of  lactic  acid  in  the  \irine  may  also  result  from 
d(!fective  transformation  of  ammonia  into  urea  by  a  diseased  liver,  tlie  acid  neu- 
tralizing, and  being  excreted  with,  the  ammonia;  in  this  case  no  defective  oxida- 
tion need  be  assumed.     However,   administration  of  phlorhizin    to    phosphorus 

'"  Harvey  Society  Lectures,  1916. 


ACIDOSTS  IN  NEPHRITIS  565 

])oi.sono(l  (logs  causes  both  ammonia  ami  lactic  acid  to  disappear  from  the  urine, 
indicating  that  the  ammonia  is  the  protective  sul)stance  which  neutralizes  the 
lactic  acid,  and  not  the  reverse.  likewise,  lactic  acid  acts  as  a  defensive  mech- 
anism when  excess,  of  alkali  is  administered,  appearing  in  the  urine  in  slightly 
increased  amounts.*' 

Sarcolaetic  acid,  which  is  dextrorotarj-,  must  be  distingiiished  from  its  optical 
isomer,  the  inactive  lactic  acid  that  is  produced  by  fermentation.  When  this  fer- 
mentation lactic  acid  is  formed  in  the  stomach  and  enters  the  blood,  it  ordinarily, 
like  other  ingested  organic  acids,  is  combined  by  the  blood  alkalies  and  oxidized 
to  carbonates.     It  is  doubtful  if  it  ever  enters  the  urine."* 

As  a  general  rule  sarcolaetic  acid  is  not  found  abundant  in  the  urine  together 
with  the  acetone  bodies,  but  is,  indeed,  antiketogenic.  Its  appearance  in  the 
urine  indicates  that  glycogen  is  not  completely  burned,  and  this  condition  is 
usually  accompanied  with  fatty  changes  in  the  liver,  which  also  depend  on  lack  of 
oxidation.  Throughout  the  clinical  forms  of  acidosis,  lactic  acid  and  fatty  degen- 
eration are  always  associated  (Ewing).  To  assume,  as  has  been  generally  done, 
that  the  lactic  acid  appears  in  the  urine  when  hepatic  alterations  are  marked,  be- 
cause of  the  loss  of  the  liver  tissue  which  should  destroy  it,  is  probably  not  warranted. 
Rather,  the  liver  conditions  and  the  formation  of  lactic  acid  depend  upon  the  same 
cause,  which  is  a  defective  oxygen  supply  or  interchange,  either  general  or  local. *^ 

Acidosis  in  Nephritis. — This  presents  a  very  different  urinary 
chemistry  from  diabetic  acidosis,  in  that  there  is  no  excessive  excretion 
of  organic  acids  or  ammonia,  and  indeed  no  other  striking  urinary 
change  to  account  for  the  acidosis  which  may  be  severe,  undoubtedly 
often  terminating  hfe.^^  There  is  a  definite  increase  in  the  inorganic 
phosphate  content  of  the  blood  (Marriott  and  Howland^^)  in  nephritis 
with  acidosis,  and  often  the  urinarj'  aciditj'  is  decreased,  so  the  acido- 
sis is  attributed  to  reduced  capacity  of  the  kidney  to  excrete  acid 
phosphates  which  is  one  of  the  most  important  normal  mechanisms  for 
maintaining  the  neutrality  of  the  blood.  The  most  marked  acidosis 
is  observed  in  those  types  of  nephritis  that  are  associated  with  uremia, 
i.  e.,  advanced  chronic  glomerulo-nephritis  and  acute  nephritis,  but 
not  in  the  chronic  parenchymatous  types.  The  nocturnal  hyperp- 
noea  of  nephritis  probably  is  the  result  of  acidosis  (Whitney).  Acido- 
sis generally  parallels  in  degree  the  impairment  in  excretory  capacity 
of  the  kidney,  and,  in  contrast  to  diabetes,  it  does  not  reach  a  high 
grade  so  long  as  the  excretion  of  acid  by  the  kidney  is  comparatively 
efficient  (Sellards).  While  administration  of  sodium  bicarbonate 
corrects  the  acidosis  it  has  little  effect  on  the  course  of  the  disease, 
or  on  the  amount  of  phosphate  in  the  blood.  With  this  high  phosphat- 
emia  there  is  a  reduction  in  the  blood  calcium,  which  ma}'  have  an 
unfavorable  influence  on  the  irritability  of  the  nervous  tissues.  Whit- 
ney believes  that  in  nephritis  with  acidosis  there  is  probably  some 
excessive  production  of  acid,  as  a  kidney  with  greatly  impaired  excre- 

"  Macleod  and  Knapp,  Ainer.  Jour.  Physiol.,  1918  (47),  189. 

**  The  theory  of  Boix  that  cirrhosis  of  the  liver  may  be  produced  bj'  butyric 
acid  formed  in  gastric  fementation  could  not  be  corroborated  by  Joannovics, 
Arch.  int.  Pharmacodyn.,  1905  (15),  241. 

83  See  Macleod  and  Wedd  (Jour.  Biol.  Chem.,  1914  (18),  446)  who  found 
that  reducing  the  oxygen  supply  to  the  liver  caused  a  marked  rise  in  the  lactic 
acid  content  of  the  hepatic  blood. 

9"  See  \Miitnev,  Arch.  Int.  Med.,  1917  (20),  931. 

91  Arch.  Int.  Med.,  1916  (IS),  708. 


566  ABNORMALITIES  IN  METABOLISM 

tory  capacity  may  be  able  to  maintain  a  normal  acid  threshhold 
Begun  and  Miinzer^^"  attribute  part  of  the  acidosis  to  a  decreased 
formation  of  NH3  in  metabolism. 

Other  Diseases  with  Acidosis. — While  a  slight  degree  of  acidosis 
undoubtedly  may  occur  in  many  conditions,  there  are  few  conditions 
in  which  it  is  of  importance.     Chief  of  these  are  the  following: 

Asiatic  Cholera  exhibits  often  a  severe  acidosis,  presumabh'-  because  of  the 
frequencj'  of  severe  renal  lesions  (Sellards^'*).  Loss  of  bases  in  the  evacuations  may 
also  be  a  factor.  The  acidosis  differs  from  that  of  nephritis  in  the  excretion  of 
large  amounts  of  ammonia,  but  usually  without  acetone  bodies.  Other  infectious 
diseases  do  not  commonly  exhibit  any  significant  degree  of  acidosis,  rheumatic 
fever  alone  excepted. 

Acidosis  in  infancy. — Both  true  acidosis  and  acetonuria,  with  or  without 
acidosis,  occur  frequently  and  easily  in  the  young.  Normalh'  the  urine  of  infants 
and  children  contains  about  3  mg.  of  acetone  per  kilo  of  body  weight,  and  may  be 
increased  to  ten  times  that  amount  by  fasting.  ^^  Also  the  amount  of  acetone 
bodies  in  the  blood  is  greater  and  more  readily  increased  by  fasting  than  in  adults. '^ 
Presumably  the  infantile  organism  has  a  lower  oxidative  capacity,  since  it  excretes 
unoxidized  organic  acids  from  relatively  slight  causes,  in  corroboration  of  which  is 
the  observation  of  Pfaundler^*  that  the  proportion  of  nitrogen  in  the  urine  of 
infants  in  forms  other  than  urea  is  higher  than  in  adults.  However,  according  to 
Howland  and  Marriott"  serious  acidosis  in  infancy  and  childhood,  although 
frequent,  is  usually  not  due  to  the  acetone  bodies.  It  is  especially  important  in 
severe  choleriform  diarrhea,  possibly  because  of  excretion  of  bases  in  the  discharges, 
and  in  burns  and  severe  nephritis,  acidosis  is  of  significance.  In  ileocolitis  true 
acetonemic  acidosis  has  been  observed. 

Terminal  Acidosis. — In  many  dying  persons  the  final  symptomatology  is 
strikingly  like  that  of  death  from  acidosis,  and  Wliitney  has  found  that  the  final 
figures  for  alkali  reserve  in  the  blood  of  animals  killed  by  acid  intoxication  are  of 
the  same  order  as  those  that  may  sometimes  be  obtained  from  dying  patients  ^^^th 
many  different  diseases.  Of  forty  cases  studied  by  him,  in  all  but  three  there  was 
marked  acidosis,  and  in  many  there  was  a  degree  of  acidosis  sufficient  of  itself  to 
cause  death.  Sellards  notes  typical  acidosis  in  advanced  atrophic  cirrhosis,  but  this 
has  not  yet  been  sufficiently  studied  to  permit  of  proper  interpretation.  Whenever 
the  blood  pressure  becomes  greatly  lowered,  as  in  shock,  there  may  occur  an  actual 
acidosis. 

Severe  anemia,  both  primary  and  secondarj^,  may  exliibit  a  moderated  degree  of 
acidosis,  but  in  only  about  one  fifth  of  all  cases  examined  by  Sellards. 

Alkalosis 

The  occurrence  of  an  increase  in  OH  ions  of  the  blood,  or  of  an 
abnormally  high  alkali  reserve  and  increased  capacity  to  carry  CO2, 
seems  to  occur  infrequently,  presumably  since  the  trend  of  metabolic 
processes  is  to  produce  acid  substances.  The  chief  example  is 
furnished  in  tetany,  whether  the  result  of  parathyroid  insufficiency  or 
pyloric  obstruction;^^  in  the  latter  case  excretion  of  acid  which  does 
not  enter  the  intestine  to  be  neutralized  might  account  for  an  excess 

»'«  Zeit.  exp.  Path.,  1919  (20),  78. 

»2  Veederand  Johnstone,  Amer.  Jour.  Dis.  Child.,  1917  (13),  89. 

"  See  Moore,  ibid.,  1916  (12),  244.  The  statement  that  there  is  usually  a 
relative  acidosis  in  newborn  infants  could  not  be  corroborated  by  Seham,  ibid. 
1919  (18),  42. 

"  Jahrb.  f.  ICinderheilk.,  1901  (54),  247. 

"  Amer.  Jour.  Dis.  Child.,  191G  (12),  459. 

««  See  Wilson  et  al.,  Jour.  Biol.  Chem.,  1915  (21),  169;  1915  (23),  89;  McCann, 
ibid.,  1918  (35),  553. 


ACETONURIA  567 

of  bases  in  the  blood.  Tetany  is  improved  by  giving  acids,  and  pre- 
sumably the  convulsions  of  the  disease  have  a  similar  (ifTcct  through 
increased  acid  production.  IVIenten^^  has  found  an  increased  OII- 
ion  concentration  in  the  blood  to  be  characteristic  of  cancer,  whether 
involving  the  stomach  or  not,  although  sometimes  observed  in  other 
conditions,  especially  diabetes  before  the  stage  of  acidosis.  A  slight 
degree  of  alkalosis  may  be  produced  by  feeding  large  amounts  of 
alkali,  in  which  case  some  of  the  alkah  is  removed  in  the  urine  com- 
bined with  lactic  acid.^"^ 

ACETONURIA  WITHOUT  MARKED  ACIDOSIS 

Not  infrequently  acetone  bodies  are  found  in  the  urine  of  patients 
suffering  from  the  most  diverse  diseases.  It  is  customary  to  refer 
to  this  condition  as  '^acetonemia"  or  " acetonuria,"  and  to  ascribe 
many  of  the  observed  symptoms  to  "acid  intoxication."  The  pres- 
ence of  these  substances  in  the  urine,  however,  is  by  no  means  evidence 
of  acidosis,  for  excretion  of  considerable  amounts  of  acetone  bodies 
may  occur  without  reduced  COa-carrying  capacity  of  the  blood  and 
they  may  be  absent  with  marked  acidosis.  In  addition,  it  must  be 
kept  in  mind  that  acidosis  may  result  from  other  causes  than  over- 
production of  organic  acids;  e.gr.,  acid  phosphate  retention  in  nephritis, 
or  loss  of  bases  from  biliary  or  pancreatic  fistula.  In  no  other  condi- 
tions do  the  amounts  of  organic  acids  in  the  urine  approximate  the 
amounts  found  in  diabetic  coma.  Therefore,  the  intoxication  in 
these  cases  is  probably  not  due  to  the  acids,  but,  on  the  contrary,  the 
presence  of  the  acetone  bodies  is  due  more  often  to  the  effects  of  toxic 
substances  of  diverse  origins  and  natures. 

Anesthesia. — As  shown  first  by  Greven  (1895),  and  especially  by  Brewer  and 
Helen  Baldwin^^  acetone  is  nearly  always  present  in  the  urine  during  the  first 
twenty-four  hours  after  administration  of  chloroform  or  ether,  and  occasionally 
diacetic  acid  appears  on  the  second  or  third  daj'  after;  but  ordinarily  there  is  no 
increase  in  organic  acids  in  the  urine.  There  is  usually  little  or  no  demonstrable 
acidosis.^  The  starvation  preceding  and  following  the  operation  is  also  a  factor 
of  considerable  importance.  It  does  not  seem  probable  that  the  s.\  mptoms 
observed  in  typical  cases  of  delayed  chloroform-poisoning  {q.  v.)  are  due  chiefly, 
if  at  all,  to  acid  intoxication  per  se,  but  rather  are  the  result  of  extensive  injury  to 
the  parenchymatous  organs,  particularly  the  liver,  by  the  chloroform,  which  causes 
a  condition  resembling  acute  yellow  atrophy  or  phosphorus-poisoning. 

Cachectic  Acetonuria. — Acetone  and  diacetic  acid,  but  less  abundantly  the 
oxybutyric  acid  are  found  in  the  urine  in  many  conditions  associated  with  wasting, 
among  which  may  be  especially  mentioned : 

Starvation. — -Acetone,  which  is  normalh*  excreted  through  the  lungs  for  the 
most  part  (80-90  per  cent,  of  that  produced)  appears  in  excess  in  the  urine  very 
soon  after  fasting  begins,  there  being  more  produced  than  can  be  exhaled.  It  is 
associated  \\'ith  diacetic  acid  and  oxybutyric  acid,  which  may  reach  10  to  20  grams 
per  day  in  starvation,  and  even  higher  figures  are  recorded.  The  urinary  ammonia 
nitrogen  runs  parallel  to   the  acid  excretion.     The  use  of  a  carbohydrate  free 

«"  Jour.  Cancer  Res.,  1917  (2)  179. 

'*  Macleod  and  Knapp,  Amer.  Jour.  Physiol.,  1918  (47),  189. 
S3  .Jour,  of  Biol.  Chem.,  190t)  (1),  239. 
1  See  Caldwell  and  Cleveland,  Surg.  Gyn.  Obst.,  1917  (25),  22. 


568  ABNORMALITIES  IN  METABOLISM 

diet  is  also  accompanied  bj^  a  marked  acetonuria,^  no  matter  how  much  fat 
is  supplied,  and  may  reach  a  point  where  several  grams  of  oxybutyric  acid  are  being 
excreted  per  day  without  symptoms  of  serious  intoxication.  A  relativel}-  small 
amount  of  carbohydrate  (80  grams)  is  sufficient  to  prevent  this  acidosis.  If  the 
meat-fat  diet  is  continued  for  some  time,  however,  there  seems  to  be  some  sort  of 
adaptation  so  that  the  acetonuria  diminishes  until  practically  normal  figures  maj^ 
be  reached. 

Pregnancy. — During  pregnancy  the  urine  usually  contains  acetone  bodies  in 
slight  excess,  and  occasionally  in  large  excess  in  women  who  are  suffering  from 
the  toxemias  of  pregnancy.  Here  there  is  a  rise  in  ammonia  far  bej'ond  the  propor- 
tion of  acetone  bodies,  partlj^  because  of  the  large  amounts  of  lactic  acid  which  are 
excreted,  and  partly  from  abnormal  protein  metabolism  and  tissue  destruction,  but 
the  proportion  of  the  urinary  nitrogen  which  is  constituted  bj'  ammonia  is  too 
inconstant  to  serve  as  a  prognostic  and  operative  guide.  Ewing  has  obsen'ed  a 
case  of  pernicious  vomiting  with  75  per  cent,  of  the  total  nitrogen  as  ammonia,  and 
no  urea, — while  there  maj^  occur  fatal  cases  without  large  excess  of  ammonia. 
Higher  ammonia  figures  are  usually  reached  in  pernicious  vomiting  of  pregnancy 
than  in  eclampsia;  in  neither  is  the  acidosis  present  sufficient  to  account  for  the 
intoxication.  (See  discussion  of  "Eclampsia.")  Even  normal  pregnant  women 
seem  to  show  a  reduced  abilitv  to  tolerate  a  deficiency  in  the  carbohydrates  of  the 
diet.  3 

Cyclic  Vomiting. — Here  the  urine  usually  shows  acetone  bodies,  lactic  acid, 
indican  in  excess,  and  a  rise  in  the  proportion  of  neutral  to  oxidized  sulphur  (How- 
land  and  Richards).  As  these  findings  may  persist  in  spite  of  absorption  of 
carbohydrates,  they  are  not  entirely  due  to  starvation,  and  there  are  severe  fatty 
changes  in  the  liver  and  kidneys,  indicating  a  toxemic  origin  associated  with  defec- 
tive oxidation.  Mellanby^  found  a  considerable  creatine  elimination  in  a  tj-pical 
case.     There  is,  however,  usually  no  acidosis,  although  it  maj'  develop. 

Inanition  and  Cachexia. — Under  this  heading  may  be  grouped  the  acetonuria 
observed  in  intestinal  disturbances  in  children,*  hysterical  vomiting,  psychoses, 
and  cancer.  In  each  of  these  conditions  coma  of  the  type  of  diabetic  coma  has 
sometimes  been  observed,  and  in  all  of  them  acetonuria  is  common,  the  reasons 
being  obvious  after  the  above  discussion.  A  relative  acidosis  may  also  result 
from  deficiency  of  bases  in  the  diet  of  gro^\ing  infants.  In  many  cases  of  acidosis  of 
infants  there  is  not  sufficient  increase  in  the  acetone  bodies  of  the  blood  to  account 
for  the  acidosis;''  on  the  other  hand,  most  of  the  children  excreting  acetone  bodies 
in  the  urine  do  not  have  acidosis. 

Retention  of  placenta  or  fetus,  acetonuria  being  considered  of  diagnostic  value 
in  determining  the  death  of  the^fetus  in  utero,'  but  not  in  extrauterine  pregnancy 
(Wechsberg).* 

In  uremia,  as  previously  mentioned,  organic  acids  may  appear  in  the  urine, 
but  apparently  as  a  result,  and  not  as  the  cause,  of  the  uremia  (Orlowski).  There 
is  usually  some  acidosis  in  advanced  nephritis,  but  marked  only  in  uremia  as  dis- 
cussed above. 

Other  Conditions. — Acetonuria  is  observed  inconstantly  in  fever,  especially  in 
children;^"  also  after  poisoning  by  many  driigs,  including,  besides  the  heavy  metals, 
morphine,  atropine,  antipyrine,  and  phlorhizin.  Pneumonia  is  accompanied  by 
acidosis,^  often  of  serious  degree,  subsiding  rapidly  after  the  crisis.  Acidosis  seems 
to  be  an  important  feature  in  gas  gangrene.  i°  At  high  altitudes  there  is  always  an 
acidosis,  which  stimulates  the  respiratory  center  to  increased  activity.     In  asphy.x- 

2  See  Higgins,  Peabody  and  Fitz.  Jour.  Med.  Pes.,  1916  (34),  263. 

3  Porges  and  Novak,  Berl.  klin.  Woch.,  1911  (48),  1757. 
^Lancet,  July  1,  1911. 

6  See  Howland  and  Marriott,  Amcr.  Jour.  Dis.  Child.,  1916  (11),  309;  (12), 
459. 

«  Moore,  Amer.  Jour.  Dis.  Child.,  1916  ri2).  244. 

^  See  Frommer,  Berl.  klin.  Woch.,  1905  (42),  1008. 

8  Wien.  klin.  Wocli.,  190{)  (19),  953. 

8"  See  Garland,  Arch.  Pediat.,  1919  (36),  468;  Veoder  and  Johnston,  Amor.  Jour. 
Dis.  Chil.,  1920  (19),  141. 

»  Lewis  and  Barcroft,  (Juart.  Jour.  Med.,  1915  (8),  108. 
»»  Wright  and  Fleming,  Lancet,  Feb.  9,  1918. 


INTOXICATION  OF  FATIGUE  oG9 

ial  conditions  of  all  sorts  more  or  less  acidosis  is  present,  c.  g.,  uncompensated 
cardiac  defects,  severe  anemia,  gas  poisoning,  surgical  or  traumatic  shock. 

FATIGUE" 

The  symptoms  of  fatigue,  whether  general  or  local,  soom  to  he  due 
to  an  intoxication  with  the  products  of  the  excessive  metabolic  activ- 
it}',  and  part  of  the  symptoms,  at  least,  seem  to  be  due  to  acid  intox- 
ication. Among  the  metabolic  products  of  muscular  activity  are 
known  to  be  creatin,  creatinin,  sarcolactic  acid,  and  carbon  dioxide. 
The  amount  of  acid  developed  in  an  active  muscle  is  quite;  consider- 
able, and  when  the  activity  is  violent  or  prolonged  the  sarcolactic 
acid  accunmlates,  being  formed  faster  than  it  can  be  removed.  Part 
of  the  acidity  of  the  muscle  is  due,  however,  not  to  the  sarcolactic 
acid  itself,  but  to  monopotassium  phosphate  (KH2PO4),  which  is 
formed  by  the  action  of  the  sarcolactic  acid  upon  the  dipotassium 
phosphate  present  in  the  blood  and  muscle.  The  effect  of  these 
various  substances  upon  muscular  fatigue  has  been  studied  experi- 
mentally, and  while  the  creatin  seems  not  to  be  a  "fatigue  substance," 
sarcolactic  acid,  monopotassium  phosphate,  hydroxybutyric  acid, 
and  carbon  dioxide  all  cause  muscle  tissue  to  react  to  stimuli  in  the 
same  way  that  a  fatigued  muscle  does  (Lee^-).  Presumably  these 
substances  act  chiefly  by  virtue  of  their  carrying  hydrogen  ions,  al- 
though there  is  some  evidence  that  the  negative  ions  of  lactic  and  oxy- 
butyric  acids  and  some  positive  ions,  especially  potassium,  are  capable 
of  producing  certain  fatigue  phenomena  (Scott).  Indole,  skatole  and 
phenol  may  also  produce  fatigue  conditions. 

It  is  quite  probable  that  the  muscular  weakness  of  diabetics,  and 
the  exhaustion  associated  with  many  conditions  in  which  organic 
acids  appear  in  the  urine  in  abnormal  quantities,  depend,  at  least  in 
part,  upon  the  efTect  of  these  acids  upon  the  muscle  tissue,  for  Lee 
found  that  /3-oxybutyric  acid  causes  the  same  fatigue  reaction  in  mus- 
cles as  does  sarcolactic  acid.  Furthermore,  sarcolactic  acid  itself 
often  appears  in  the  urine  in  these  conditions.  It  may  be  added  that 
in  fatigued  animals  the  alkalinity  of  the  blood  (by  titration)  has  been 
found  decreased  (Geppert  and  Zuntz),  and  the  proportion  of  the 
urinarj^  nitrogen  that  appears  in  other  combinations  than  urea  is 
increased  (Poehl).^^  Fatigue  in  man  causes  an  increased  urinary 
acidity. -^^^ 

The  "Toxins"  of  Fatigue. — In  extreme  exhaustion  the  evidences 
of  a  general  intoxication  often  become  severe,  so  that  the  condition 
may  resemble  an  acute  febrile  disease  and  last  for  several  days.     It 

"  Full  bibliography  by  Spaeth,  Jour.  Indust.  Hyg.,  1919  (1),  22. 

1^  Jour.  Amer.  Med.  Assoc,  1906  (46),  1491;  where  is  given  a  complete  review 
of  the  subject  of  fatigue,  with  the  literature.  Also  see  Scott,  Public  Health  Re- 
ports, 1918  (33),  605. 

"  Deut.  med.  Woch.,  1901  (27),  796. 

13"  Hastings,  Public  Health  Rep.,  1919  (34),  1682;  Barach,  .^ler.  Jour.  Med. 
Sci.;  1920  (159),  398. 


570  ABNORMALITIES  IN  METABOLISM 

seems  very  probable  that  substances  more  toxic  than  the  above- 
mentioned  acids  are  involved.  Weichardt^*  claimed  that  he  had  de- 
monstrated a  toxic  substance,  produced  by  muscular  fatigue,  which 
in  structure  resembles  the  bacterial  toxins,  called  by  him  kenot  ox  in, ^^ 
and  against  which  an  antitoxin  may  be  obtained.  This  toxic  material 
is,  he  believes,  formed  from  the  protein  molecule  in  the  first  stages  of 
its  decomposition,  as  a  side  product  which  is  normally  protected  against 
by  a  formation  of  an  antitoxin,  rather  than  by  being  split  up  further, 
as  is  the  case  with  the  rest  of  the  protein  molecule.  The  study  of 
anaphylaxis  has  led  to  so  many  evidences  of  the  remarkable  toxicity 
of  the  products  of  protein  cleavage,  that  the  possibility  that  some  of 
these  may  be  responsible  for  fatigue  cannot  be  entirely  disregarded 
at  present,  ^^  although  Lee  and  Aronovitch^^  could  not  demonstrate 
toxic  substances  in  fatigued  muscles,  and  no  one  has  been  able  to 
confirm  Weichardt's  specific  findings.  The  following  observation  of 
Mosso  indicates  that  the  blood  of  fatigued  animals  contains  toxic 
substances:  If  blood  is  transfused  from  an  exhausted  dog  to  a  normal 
dog,  from  which  an  equivalent  amount  of  blood  has  been  withdrawn, 
this  second  dog  will  show  the  usual  manifestations  of  fatigue.  Men- 
denhalP^  has  found  that  the  heart  is  also  affected  by  the  products  of 
muscular  fatigue.  Recent  studies  in  shock  (Bayliss,  Cannon)  attrib- 
ute the  manifestations  of  shock,  at  least  in  part,  to  toxic  cleavage 
products  of  injured  tissues,  and  many  of  these  manifestations  are 
allied  to  fatigue. 

Mental  Fatigue. — The  chemical  changes  of  mental  fatigue  are  not 
kno'wn,  but  the  ganglion-cells  show  marked  structural  alterations 
as  a  result  of  fatigue,  chromatolysis  often  being  very  striking.  Since 
lecithin  forms  so  important  a  part  of  the  nervous  system,  it  is  tempting 
to  imagine  that  in  fatigue  excessive  quantities  of  its  toxic  decomposi- 
tion-product, choline,  and  the  still  more  toxic  derivative  of  choline, 
neurine,  are  formed  in  considerable  amounts  and  cause  part,  at  least, 
of  the  intoxication. 

That  choline  or  neurine  actually  are  the  cause  of  any  of  the  symp- 
toms of  fatigue,  however,  has  not  been  established;  but  Donath^^ 
considers  choline  an  important  factor  in  the  production  of  epileptic 
convulsions ^^  Animals  kept  for  a  long  time  from  sleeping  are  said  to 
show  the  presence  in  their  blood,  cerebro-spinal  fluid  and  brain  tissues, 
of  a  poisonous  property  causing  somnolence  in  other  animals  (Legendre 

'■'  "  Ueber  Ermiidungsstoffe,  "  Enke,  Stuttgart,  1912;  KoUe  and  Wassermann's 
Handbuch,  1913  (2),  1499. 

"*  Hec  Woichardt  and  Schwenk,  Zeit.  physiol.     Chcm..  1913  (83),  381. 

"  The  failure  of  various  investigators  to  corroborate  \A'cichardt  is  discussed  by 
Konrich,  Zeit.  f.  Hyg.,  1914  (78),  1;  Ivorff- Petersen,  ibid.,  p.  37. 

1'  Proc.  Soc.  Exp.  Biol.  Med.,  1917  (14),  153. 

»«  Amer.  .lour.  Physiol.,  1919  (48),  13. 

"•  Zeit.  phy.siol.  Chem.,  1903  (39),  52(5. 

'"  Concerning  the  theories  and  literature  of  the  subject  of  epilepsy  in  relation 
to  its  pathologieal  chemistry  and  to  autointoxication,  sec  the  review  of  Masoin. 
Arch,  internat.  de  Pharmacodynamie,  1904  (13),  387.  ' 


POISONS  OF  BURNS  571 

and  Pi^ron).'^  This  cannot  well  be  choline  or  any  similar  substance, 
for  it  does  not  filter,  is  insoluble  in  alcohol,  and  is  destroyed  by  heating 
at  65°. 

THE  POISONS  PRODUCED  IN  SUPERFICIAL  BURNS" 

In  a  certain  proportion  of  cases  of  extensive  but  superficial  burns, 
death  follows  after  an  interval  of  from  six  hours  to  a  few  days, 
apparentl}^  because  of  a  profound  intoxication.  As  evidence  of  intoxi- 
cation we  have  not  only  clinical  manifestations,  such  as  delirium,  hemo- 
globinuria, and  albuminuria,  vomiting,  bloody  diarrhea,  etc.,  but,  more 
convincingly,  the  anatomical  findings  at  autopsy,  which  are  strikingly 
similar  to  those  resulting  from  acute  intoxication  with  bacterial  prod- 
ucts. Bardeen  found  quite  constantly  cloudy  swelling  and  focal  and 
parenchymatous  degeneration  in  the  liver  and  kidneys;  softening  and 
enlargement  of  the  spleen  with  focal  degeneration  in  the  Malpighian 
bodies;  and  particularly  degenerative  changes  in  the  Ij-mph-glands 
and  intestinal  follicles  resembling  those  observed  in  diphtheria,  which 
McCrae-'  considers  due  to  proliferation  and  phagocytosis  by  the  en- 
dothelial cells  of  the  lymphatic  structures.  The  severe  degenerative 
changes  seen  in  the  adrenals  and  myocardium  especially  recall  diph- 
theria intoxication  (Weiskotten).-'*  Marked  changes  are  usually 
present  in  the  blood,  consisting  of  fragmentation  and  distortion  of  the 
red  corpuscles,  hemoglobinemia,  loss  of  water  with  a  relative  increase 
in  the  number  of  corpuscles  by  from  one  to  four  millions  per  cubic 
millimeter,  an  increase  in  the  blood  platelets,  and  a  rise  in  the  number 
of  leucocytes  as  high  as  30,000  to  50,000.-^  Hemoglobinuria  is  also 
frequently  present,  and  almost  constantly  gastrointestinal  irritation 
occurs,  with  anatomical  evidences  of  acute  enteritis,  acute  gastritis, 
and  occasionally  gastric  or  duodenal  ulcers.  According  to  Korolenko,-^ 
the  sjnnpathetic  nervous  system  is  seriously  involved. 

It  therefore  seems  probable  that  poisons  are  formed  as  a  result 
of  superficial  burns,  which  have  the  effect  of  causing  hemolysis,  and 
which  are  also  cytotoxic  for  parenchymatous  cells.  These  hypotheti- 
cal poisons  seem  to  be  eliminated  In'  the  intestines  and  kidneys,  which 
are  injured  by  the  poisons  in  their  passage  through  these  organs.  The 
attempts  to  explain  all  the  observed  effects  of  burns  as  due  to  throm- 
bosis or  to  embolism  by  altered  corpuscles  seem  to  have  failed,  for 
the  peculiar  location  of  the  lesions  (e.  g.,  duodenal  ulcers,  necrosis  in  the 

21  Zeit.  allg.  Physiol.,  1912  (14),  2.35. 

"Literature  given  by  Bardeen,  Johns  Hopkins  Hosp.  Reports,  1898  (7),  1.37; 
Eyff,  Cent.  Grenzgeb.  Med.  u.  Cliir.,  1901  (4),  428;  Pfeiffer,  Virchow's  Arch., 
190.5  (180),  367.  Full  discussion  of  theories  by  Vogt,  Zeit.  exp.  Path.  u.  Pharm., 
1912  (11),  191. 

"  Amer.  Med.,  1901  (2),  735. 

"Jour.  Amer.  Med.  Assoc,  1917  (69),  776;  1919  (72),  259;  also  Xakata,  Corr 
Bl.  Schw.  Aerzte.  1918  (48),  1283. 

"  Locke.  Boston  Med.  and  Surg.  Jour.,  1902  (147),  480. 

-*Cent.  f.  Path.,  1903  (10),  663. 


572  ABNORMALITIES  IN  METABOLISM 

Malpighian  bodies  of  the  spleen,  etc.)  does  not  agree  with  this  hypo- 
thesis, and  there  are  too  many  evidences  of  the  presence  of  some  de- 
cidedly toxic  substance  in  the  blood.  There  can  be  no  question  that 
the  poisonous  substance  or  substances  are  formed  in  the  burned  area, 
and  not  in  the  internal  organs  as  a  result  of  hyperpyrexia,  as  shown  by 
numerous  observations.  Thus,  if  the  burned  area  is  removed  im- 
mediately (in  narcotized  experimental  animals),  death  will  be  pre- 
vented, whereas  if  the  burned  tissue  is  permitted  to  remain  for  a  few 
hours,  death  will  occur.  If  the  burned  skin  is  transplanted  to  a  normal 
animal,  this  animal  will  develop  symptoms  of  intoxication,  while  the 
burned  animal  may  be  saved  by  the  transplantation  (Vogt).  The 
poison  appears  to  be  absorbed  from  the  burned  area  into  the  blood, 
for  if  the  circulation  is  shut  off  from  the  burned  area,  no  intoxication 
results;  this  probably  explains  in  part  why  deep  destructive  burns  of 
small  areas,  which  are  associated  with  local  thrombosis,  are  much  less 
serious  than  a  superficial  slight  scalding  over  a  large  area.  Apparently 
the  poison  is  produced  chiefly  or  solely  in  the  skin,  for  burning  of 
muscle  is  not  followed  by  intoxication  (Eijkman  and  Hoogenhu^'ze).-'' 
When  one  of  a  pair  of  animals  united  to  another  by  operative  pro- 
cedure (parabiosis)  is  burned,  the  other  animal  may  become  intoxi- 
cated, while  the  intoxication  of  the  burned  animal  is  less  than  it 
would  be  if  it  were  alone  (Vogt). 

Numerous  investigators  have  reported  finding  poisonous  sub- 
stances in  the  blood,  tissues,  or  urine  of  burned  men  and  animals,  but 
the  reports  disagree  widely  in  details.-^  Thus  Dietrichs  states  that 
the  blood  of  burned  animals  contains  hemolysins  and  hemagglutinins, 
which  could  not  be  corroborated  by  Burkhardt-^  or  by  Pfcift'er.^'^ 
The  latter,  however,  finds  that  the  urine,  serum,  and  organs  of  burned 
animals  contain  substances  poisonous  for  the  same  and  for  different 
species,  which  is  in  accord  with  the  results  of  numerous  earlier  inves- 
tigators. The  poisons,  according  to  Pfeiffer,  are  neurotoxic  and  necro- 
genic  in  their  properties,  and  act  without  a  period  of  incubation; 
they  are  rapidly  weakened  on  standing  in  solution  and  by  the  action 
of  sunlight,  are  absorbed  from  the  gastro-intestinal  tract,  are  soluble 
in  water,  alcohol,  and  glycerol,  but  not  in  chloroform  or  ether,  are 
precipitated  by  HgClc  in  acid  solution,  and  by  phosphotungstic  acid, 
and  they  are  not  volatile.  Apparently,  according  to  Pfeiffer,  they 
are  not  ptomains,  nor  yet  pyridine  derivatives,  as  many  investigators 
have  contended,  but  resemble  more  closely  the  labile  poisons  of  snake 
venom,  and  have  effects  similar  to  the  unknown  poisons  that  are  con- 
cerned in  uremia.     The  neurotoxic  substance  is  more  thormoslable 

"  Virchow's  Arch.,  1906  (183),  377. 

^'^  Ravenna  and  Minassian  (Rcf.  in  Bioclicni.  Contr.,  1903  (1),  348)  state  tliat 
blood  heated  outside  the  body  to  55°-G0°  is  toxic,  and  causes  the  same  anatomical 
changes  as  docs  death  from  burning,  which  finding  is  corroborated  by  Helstcd. 
Arch.  klin.  Chir.,  1906  (79),  414. 

"  Arch.  klin.  Chir.,  1905  (75),  845. 

'«  Virchow's  Arch.,  1905  (180),  367;  Zeit.  f.  II vg..  1906  (54),  419. 


POrSOXS  OF  BURNS  573 

than  tho  nccrop;onic  suhstanco,  whicli  is  very  easily  destroj'od  by  heat. 
Pfciffcr  behoves  it  probable  that  the  poisons  arc  derived  from  the 
cleavage  of  proteins  altered  in  composition  by  burning,  and  he  finds 
an  enzyme  splitting  glyc3'ltryptoj)hanc  in  the  blood  and  urine  of 
burned  animals.-'^  The  hemolysis  ho  attributes  to  direct  injury  of 
the  blood  in  its  passage  through  the  heated  area,  and  not  to  the  action 
of  poisons;  this  is  very  possible,  since  red  corpuscles  fragment  after 
being  heated  to  52°,  and  may  be  seriously  impaired  functionally  at 
45°.  There  are  many  authors,  indeed,  who  consider  the  blood  changes 
the  chief  cause  of  death,  but  the  weight  of  evidence  is  in  favor  of  tho 
theory  of  the  development  of  toxic  substances  in  the  burned  skin. 

Kutscher  and  Heyde^-  believe  methyl  guanidine  to  be  the  toxic 
substance  eliminated  in  the  urine,  stating  that  it  produces  effects  sim- 
ilar to  that  caused  by  injections  of  tho  toxic  urine  from  burn  cases. 
These  symptoms  are  quite  similar  to  those  characteristic  of  anaphy- 
laxis, and  Heyde  states  that  small  burned  areas  sensitize  an  animal 
to  later  injections  of  extracts  of  burned  tissue.  He,  as  well  as  Vogt, 
are  therefore  inclined  to  believe  that  some  cases,  especially  those  dy- 
ing unexpectedly  12  or  13  days  after  the  burning,  may  be  the  result 
of  anaphylactic  reaction  to  proteins  made  of  foreign  character  by  the 
heat.^^  The  newer  observations  concerning  the  presence  of  toxic  sub- 
stances in  the  urine  during  anaphylactic  intoxication  are  in  harmony 
with  the  findings  in  burn  cases, ^■^  although  the  identity  of  method 
guanidine  with  the  toxic  agent  is  questionable. 

Burn  Blisters. — -The  contents  of  burn  blisters  resemble  the  fluid  of 
inflammatory  edemas  generally.  K.  Mornor^^  found  5.031  per  cent, 
of  proteins,  which  included  1.359  per  cent,  of  globulin  and  0.011 
per  cent,  of  fibrin;  there  was  also  present  a  substance  reducing  cop- 
per oxide,  but  no  pyrocatechin. 

31  Zeit.  Immunitiit.,  1915  (23),  473. 

32  Cent.  f.  Phvsiol.,  1911  (25),  441. 

"  Heyde,  Med.  Klinik,  1912  (S),  263. 

3^  See  Pfeiffer,  Zeit.  Immunitat.,  1913  (18),  75. 

35  Skand.  Arch.  Physiol.,  1S95  (5),  272. 


CHAPTER  XXI 

GASTRO-INTESTINAL  "AUTOINTOXICATION"  AND 
RELATED   METABOLIC  DISTURBANCES 

Under  this  heading  are  commonly  included  all  intoxications  that 
can  be  ascribed  to  the  absorption  from  the  gastro-intestinal  tract  of 
toxic  substances  that  have  been  formed  within  its  contents,  either 
by  the  action  of  the  digestive  ferments  or  of  putrefactive  bacteria. 
The  propriety  of  considering  such  conditions  as  examples  of  auto- 
intoxication is  properly  questioned,  since  it  is  often  difficult  to  deter- 
mine whether  the  putrefaction  occurred  within  the  body,  or  had 
already  taken  place  in  the  food  before  it  was  eaten.  But  even  those 
who  would  limit  the  use  of  the  term  autointoxication  to  intoxication 
with  the  products  of  cellular  metabolism,  must  admit  the  possibiUty 
of  products  of  metabolism  reentering  the  blood  from  the  contents  of 
the  bowels  through  the  intestinal  wall,  since  the  bile,  and  perhaps 
also  the  intestinal  juice,  contain  excrementitious  substances  which 
may,  in  case  of  defective  fecal  elimination,  be  reabsorbed  into  the 
blood.  Therefore,  in  gastro-intestinal  disturbances  we  have  the  pos- 
sibility of  both  true  autointoxication  and  intoxication  by  putrefactive 
products  occurring  together  in  an  inseparable  way,  and  the  usual 
inclusion  of  gastro-intestinal  intoxication  in  the  discussion  of  auto- 
intoxication would  seem  to  be  justifiable  as  well  as  expedient. 

The  possible  sources  of  poisonous  substances  arising  in  the  gastro- 
intestinal tract  are  numerous.  They  may  be  formed  either  from  the 
food-stuffs,  or  from  the  secretions  and  excretions  of  the  body  that 
enter  the  alimentary  canal;  and  they  may  be  formed  either  by  the 
digestive  ferments  or  by  the  bacteria  of  the  intestinal  contents.  Hence 
the  number  of  these  products  is  enormous,  and  we  are  by  no  means  sure 
that  those  that  have  yet  been  identified  include  the  most  important  or 
most  toxic;  furthermore,  at  the  present  time  we  are  far  from  sure  that 
any  of  these  materials,  known  or  unknown,  are  important  factors  in 
human  pathology.  To  classify  the  poisonous  substances  that  arc  known 
to  be  formed  in  the  alimentary  canal,  and  which  might,  under  certain 
conditions,  cause  an  intoxication,  is  extremely  difficult,  because  of 
the  uncertainty  of  our  information;  but,  using  as  a  basis  the  sources 
of  tile  substances,  they  may  be  classified  as  follows:^ 

^  Modified  from  Weintraud,  Ergeb.  allg.  Pathol.,  1897  (4),  1,  who  gives  ex- 
haustive discussion  and  bibliography  to  that  date. 

674 


PROTEOSES  AND  PEPTONES  575 

I.  The  consdtuents  of  the  digestive  secretions,  including  the  Inlc  salts  and 
pigments,  pepsin,  and  trypsin. 

II.  Products  of  normal  digestion: 

(a)  From  proteins — proteoses,  peptones,  amino-acids. 

(b)  From  fats — fatty  acids  and  glycerol. 

III.  Products  of  putrefaction  and  fermentation: 

(a)  From  proteins: 

(1)  From  the  aromatic  radicals  (tyrosine,  phenylalanine,  trypto- 
phane)— indole,  skatole,  skatole-carbonic  (or  indole-acctic)  acid, 
phenol,  crcsol,  dioxyphenols,  and  the  pressor  bases. 

(2)  From  the  fatty  acid  radicals — fatty  acids  (especially  butyric  and 
acetic),  acetone,  ammonia,  amino-acids,  carbon  dioxide,  hydro- 
gen, marsh-gas.  Also  ptomains;  cadaverine,  putrescine,  ethyli- 
dendiamine,  isoamylamine. 

(3)  From  the  sulphur-containing  radicals — H2S,  methyl  mercaptan, 
ethyl  mercaptan,  ethyl  sulphid. 

(6)   From  carbohydrates: 

Fatty  acids,  the  following  having  been  detected — formic,  acetic, 
propionic,  butyric,  valerianic,  lactic,  oxybutyric,  and  succinic; 
also  acetone,  CO2,  CH4,  H2. 

(c)  From  fats: 

Higher  fatty  acids,  as  well  as  butyric  acid ;  also  glycerol.  From 
lecithin — choline,  neurine,  and  muscarine-like  bodies. 

IV.  Synthetic  products  of  bacterial  activity  (e.  q.,  botulismus)  which  cannot 
properly  be  considered  as  causing  "autointoxication." 

I.     THE  CONSTITUENTS  OF  THE  DIGESTIVE  FLUIDS 

These  call  for  but  brief  consideration,  for,  although  many  of  them 
are  known  to  be  toxic,  yet  there  is  no  evidence  that  they  cause  auto- 
intoxication, either  in  health  or  disease.  Both  pepsin  and  trypsin, 
especially  the  latter,  are  decidedly  toxic  when  injected  experimentally 
into  the  blood  (see  Enzymes),  but  they  do  not  appear  ever  to  pass 
through  the  intestinal  wall  in  sufficient  quantity  to  cause  harm,  al- 
though minute  traces  may  appear  in  the  urine;  this  harmlessness 
probably  depends  largely  on  the  known  inhibiting  action  of  the  blood 
upon  enzymes. 

The  bile  salts  are  also  toxic,  especially  hemolytic,  but  those  that  are 
reabsorbed  from  the  intestines  are  taken  back  into  the  liver  and  re- 
excreted.  This  protective  arrangement  seems  to  be  sufficient  for  all 
emergencies.  The  bile-pigments  become  converted  into  urobilinogen 
through  reduction,  and  this  is  largely  absorbed  and  eliminated  as 
urobilin.  Icterus  and  cholemia  do  not  seem  ever  to  be  produced  by 
absorption  of  bile-pigments  and  bile  salts  from  the  intestines. 

II.     PRODUCTS  OF  NORMAL  DIGESTION 

Proteoses  and  Peptones. — Under  normal  conditions,  these  are 
broken  up  in  the  intestinal  wall  into  the  amino-acids,  through  the 
agency  of  erepsin,  and  do  not  appear  in  the  blood  in  appreciable 
quantities.  To  be  sure,  certain  authors  claim  to  have  found  albumose 
in  normal  blood,  but  if  present  the  amounts  are  extremely  minute. 
In  conditions  in  which  ulceration  or  other  lesions  are  present  in  the 
gastro-intestinal  tract  it  is  possible  to  find  small  amounts  of  proteoses 
in  the  urine,  probably  absorbed  through  the  abnormal  areas,  but 
not  in  quantities  sufficient  to  account  for  any  appreciable  intoxication, 


576  GASTRO-INTESTINAL  "AUTOINTOXICATION" 

although  proteoses  are  distinctly  toxic.  This  last  statement  has  been 
much  contested,  because  the  difficulty  of  purifying  proteoses  ob- 
tained from  ordinary  sources  has  left  open  the  possibility  that  such 
toxic  effects  as  have  been  observed  are  due  to  contaminating  sub- 
stances, such  as  histamine,  and  not  to  the  proteoses  themselves.  More 
recent  work,  however,  particularly  that  of  Underhill,-  Gibson'  and 
Zunz,*  seems  to  have  established  affirmatively  the  toxicity  of  proteoses, 
whether  from  animal  or  vegetable  proteins.  Besides  the  classical 
effect  of  inhibiting  the  coagulation  of  the  blood,  the  proteoses  have  a 
lymphagogue  effect  (Heidenhain),^  cause  a  marked  febrile  reaction,^ 
and  in  doses  of  some  size  are  fatal  to  experimental  animals  (rabbits 
being  much  less  susceptible  than  dogs  and  many  other  animals). 
Locally  they  cause  a  mild  inflammatory  reaction,  which  is 
followed  by  the  appearance  of  much  connective-tissue  formation.^ 
Long  continued  injection  of  proteoses  does  not  produce  visceral 
lesions.*  The  careful  sfudies  of  Zunz  show  that  intravenous  injection 
of  hetero-albumose,  thio-albumose,  deutero-albumose  and  proto- 
albumose  cause  a  rise  in  blood  pressure,  but  large  doses  may  cause  a 
fall  in  pressure;  the  abiuret  products  of  tryptic  digestion  are  much  more 
actively  depressor  than  the  albumoses.  As  a  general  rule,  however, 
it  has  been  observed  that  the  first  products  of  protein  hydrolj^sis  are 
the  most  toxic,  and  with  further  cleavage  the  toxicity  lessens  and 
finally  disappears,  as  shown  especially  in  the  studies  on  anaphylaxis 
and  anaphylatoxin  formation.^     Thus  Wolf^°  found  that  the  amino- 

2  Amer.  Jour.  Physiol.,  1903  (9),  345;  Jour.  Biol.  Chem.,  1915  (22),  443  (litera- 
ture). See  also  Hanke  and  Koessler  on  the  relation  of  histamine  to  peptone  shock. 
Jour.  Biol.  Chem.,  1920. 

3  Philippine  Jour.  Sci.,  1914  (9),  499. 

*  Arch,  inter nat.  physiol.,  1911  (73),  110. 

5  See  also  Nolf,  Arch,  inter  nat  de  Physiol.,  1906  (3),  343. 

*  Gibson  finds  that  carefully  purified  proteoses  have  but  a  slight  pvrogenic  ef- 
fect.     (Philippine  Jour.  Sci.,  1913  (8),  475.) 

'  In  a  paper  appearing  in  the  Transactions  of  the  Chicago  Pathological  Society, 
1903  (5),  240,  I  published  the  observation  that  repeated  injections  of  Witte's 
"peptone"  (which  consists  chiefly  of  proteoses)  into  rabbits  led  to  the  production 
of  marked  cirrhosis  of  the  liver,  and  suggested  the  possibility  that  proteoses 
escaping  through  a  diseased  gastric  or  intestinal  wall  into  the  blood  might  be 
a  factor  in  the  production  of  cirrhosis  in  num.  Subsequent  observations,  how- 
ever, have  shown  that  repeated  injection  of  almost  any  foreign  protein  material 
(e.  g.,  emulsions  of  organs,  foreign  blood,  etc.,  used  in  immunization  experiments) 
will  cause  a  similar  cirrhosis  in  rabbits,  which  animals,  indeed,  often  spontaneously 
show  this  condition  when  apparently  otherwise  normal.  " Peptone"  injections  in 
dogs  and  guinea-pigs  have  tailed  to  cause  a  similar  cirrhosis,  and  hence  the  value 
of  these  and  all  other  rabbit  experiments  on  cirrhosis  of  the  liver  is  very  question- 
able; however,  the  possibility  of  the  correctness  of  the  original  conclusions  still 
remains  open. 

»  Wool  ley  e/ aZ.,  Jour.  Exp.  Med.,  1915  (22),  114.  Boughton  describes  acute 
degenerative  changes  from  Witte's  peptone.     (Jour.  Immunol.,   1919  (4),  381.^ 

"  The  statement  of  v.  Knafii-Lcnz  (Arch.  exp.  Patii.  u.  Pharm.,  1913  (73), 
292)  that  the  toxicity  of  the  cleavage  products  varies  directly  with  their  trypto- 
phane content  could  not  be  corroborated  by  Underhill  and  llendrix,  Jour.  Biol. 
Cliem.,  1915  (22),  443. 

'»  Jour,  of  Physiol.,  1905  (.32),  171. 


ALBUMOSURIA  577 

acids  do  not  cause  a  fall  of  blood  pressure,  nor  do  polypeptids.^^ 
Proteoses  have  little  if  any  power  to  stimulate  antibody  formation 
(see  Antigens,  Chap.  vii).  Whipple'-  has  described  the  isolation  of 
highly  toxic  proteoses  from  the  contents  of  closed  intestinal  loops, 
and  injection  of  these  proteoses  causes  a  marked  increase  in  nitrogen 
elimination,  presumably  from  toxicogenic  destruction  of  tissue  pro- 
teins. With  this  is  a  great  increase  in  the  non-protein  nitrogen  of  the 
blood,  partly  due  to  renal  injury.  He  also  observed  an  increased 
resistance  of  the  animals  to  these  proteoses  after  repeated  injections. 
"Albumosuria."^^ — If  proteoses  enter  the  blood  stream  they  ap- 
pear in  large  part  in  the  urine,  indicating  that  the  tissues  do  not  read- 
ily utilize  them  in  this  forni.^-'  Consequently,  when  proteoses  are  pro- 
duced in  considerable  amounts  by  autolysis  of  pathological  tissues 
they  appear  in  the  urine,  and  their  presence  is  considered  to  be  of  diag- 
nostic value. ^^  True  peptone  seems  rarely,  and  according  to  many 
observers  never,  to  appear  in  the  urine;  but  in  view  of  the  observa- 
tions that  polypeptids  often  appear  in  the  urine, ^^  it  is  probable  that 
true  peptones  also  do.  Albumoses,  therefore,  may  be  found  in  the 
urine  whenever  any  considerable  amount  of  tissue  or  exudate  is  being 
autolyzed  and  absorbed,  and  it  has  been  found  in  the  following  con- 
ditions: Suppuration  of  all  kinds;  resolution  of  pneumonia;  involu- 
tion of  the  puerperal  uterus;  carcinoma  (tw^o-thirds  of  all  cases — -Ury 
and  Lihenthal),  and  other  mahgnant  growths;  febrile  conditions  with 
tissue  destruction  (37.5  per  cent,  of  all  cases,  Morawitz  and 
Dietschy);!^  acute  yellow  atrophy,  phosphorus  poisoning,  and  eclamp- 
sia; leukemia,  especially  under  x-ray  treatment;  absorption  of  simple 
and  inflammatory  exudates;  ulcerating  pulmonary  tuberculosis,^^  and 
after  tubercuhn  reactions  (Deist). ^^  Albumosuria  is  present  in  small- 
pox and  may  serve  in  differential  diagnosis. -°  In  ulcerative  condi- 
tions of  the  ahmentary  canal  albumoses  may  be  absorbed  unchanged 
and  cause  alimentary  albumosuria.  The  normal  kidney  seems  to  be 
impermeable  to  the  small  amounts  of  proteose  that  may  be  present  nor- 
mally in  the  blood,  or  even  after  large  oral  ingestion  of  proteoses,  but 
in  parenchymatous  nephritis  it  may  escape  in  the  urine  (Henderson, ^i 
Pollak).i3 

"  Haliburton,  ibid.,  1905  (32),  174. 

12  Jour.  Exp.  Med.,  1917  (25),  461;  1918  (28),  213;  1918  (29)  397. 

13  Critical  review  given  by  PoIIak,  Zeit.  exp.  Med.,  1914  (2),  314. 

"  They  may  be  partly  hydrolyzed  into  smaller  complexes,  however,  pnmary 
proteoses  being  partly  changed  to  deutero-proteoses,  and  the  latter  partly  to 
peptones  (Chittenden,  Mendel,  and  Henderson,  Amer.  Jour.  Physiol.,  1899  (2), 
142). 

i»  See  Yarrow,  Amer.  Med.,  1903  (5),  452;  Ury  and  Lilienthal,  Arch.  f.  Ver- 
dauungskr.,  1905  (11),  72;  Senator,  International  Clinics,  1905  (4,  senes  14),  85. 

'«  Chodat  and  Kummer,  Biochem.  Zeit.,  1914  (65),  392. 

"  Arch.  f.  exp.  Path.  u.  Pharm.,  1905  (54),  88. 

18  See  Parkinson,  Practitioner,  1906  (76),  219. 

1'  Beitr.  z.  klin.  Tuberk.,  1912  (23),  547. 

2"  Primavera,  Gaz.  Int.  Med.  e  Chir.,  1913,  No.  10. 

21  Lancet,  Mar.  6,  1909. 

37 


578  GASTRO-INTESTINAL  "AUTOINTOXICATION" 

It  is  possible  that  some  of  the  symptoms  of  these  conditions  are 
due  to  intoxication  with  proteoses,  for  0.07  to  0.1  gram  deutero-albu- 
mose  will  cause  a  febrile  reaction  in  a  healthy  man,--  but  probably 
their  amount  is  usually  too  small  to  cause  appreciable  effects.--'  It  is 
well  known,  however,  that  the  characteristic  rise  of  temperature  fol- 
lowing the  injection  of  tuberculin  into  tuberculous  individuals  is  also 
produced  if  minute  quantities  of  proteose  solutions  are  injected  in 
place  of  tuberculin;  therefore,  proteoses  arising  from  autolysis  in  tu- 
berculosis may  be  of  importance  in  causing  fever  and  other  symp- 
toms.^'* Tuberculous  animals  are  said  to  succumb  to  a  much  smaller 
dose  of  deutero-albumose  than  normal  animals.-^ 

The  so-called  "Bence-Jones  albumose"  that  appears  in  the  urine 
of  patients  with  multiple  bone-marrow  tumors  is  not  a  true  albumose, 
but  is  more  closely  related  to  the  simple  proteins,  and  is  discussed 
under  the  head  of  "Chemistry  of  Tumors." 

III.     PRODUCTS  OF  PUTREFACTION  AND  FERMENTATION^^ 

We  may  perhaps  gain  some  appreciation  of  the  enormous  amount 
of  bacterial  action  that  goes  on  in  the  normal  intestinal  digestive 
processes  by  considering  the  fact  that  as  much  as  one-third  of  the  total 
weight  of  the  solids  of  normal  feces  maj^  consist  of  bacteria  (Stras- 
burger),  their  proportion  being  increased  in  diarrheal  disorders  and 
decreased  in  constipation.  They  attack  all  food-stuffs,  and  among 
the  decomposition-products  formed  through  their  activitj''  are  un- 
doubtedly many  of  considerable  toxicity.  Most  of  the  products  of  in- 
testinal putrefaction  that  have  as  yet  been  isolated  are,  however,  not 
extremely  poisonous;  but  many  of  them  are  toxic  to  some  degree,  and 
their  long-continued  absorption  may  possibly  lead  to  serious  distur- 
bances. Considering  them  first  according  to  their  origin  and  chemical 
nature,  we  take  up  first  the  products  of: 

A.  Protein  Putrefaction 

(1)  Substances  Derived  from  the  Aromatic  Radicals  of  the 
Protein  Molecule 

In  the  protein  molecule  are  contained  the  following  ainino-acids  with  an  aro- 
matic nucleus: 

NH, 


Tyrosine,  H0<^     ^CHa— CH— COOH 


"Sec  Matthes,  Arch,  cxper.  Path.  u.  Pliarin.,  1895  (30),  437. 

"  In  a  series  of  unpublislicd  e.Kperiments  I  was  unable  to  cause  amyloid  de- 
generation in  rabbits  \)y  protracted  intoxication  with  proteose  solutions. 

^''  Simon,  Arch.  exp.  Med.,  1903  (49),  449.  Concerninjj;  relation  of  tuberculin 
to  proteoses  see  review  bj'  .folles  in  Ott's  "Chemische  Pathol,  dor  Tuberculosc." 
.^M\irchlioim  and  Tuczek.  .Vrcli.  exj).  Path.  u.  I'harm.,  1914  (,77),  3>s7. 

-"  (Joiii])lctL'  liil>li()frraphv  ^ivcn  in  the  rrsuiiir  on  "Intestinal  Putrefaction"  by 
Gcrhardt,  Ergcbnisse  der  Physiol.,  1904  (111,  Abt.  1),  107.  Chemistry  of  Putre- 
faction is  reviewed  by  EUinger,  ibid.,  1907  (0),  29. 


PRODUCTS  OF  PUTREFACTION 

NH2 

Phenylalanine, 

^~\  CHj— CH— COOH 

1 

Tryptophane, 

/      >— C— CM,— CII— COOH 
\            CH 

11 

579 


In  the  intestinal  contents  have  been  found  a  number  of  substances  that  are 
undoubtedly  derived  from  these  aromatic  radicals.     They  are  (1)  phenol, 


yon 

which  is  formed  in  small  quantities,  presumably  from  tyrosine,  as  also    is  the 
closely  related  (2)  paracresol, 

and  also  (3)  ■para-oxyphenyl  acetic  acid, 

HO  <^N  CH2— COOH 
and  (4)  para-oxyphenyl-propionic  acid. 

}IO^~y  CH2— CH2— COOH 

From  the  tryptophane  are  formed  numerous  important  substances,  as  follows: 

NH2 

/-\  I 

<       >— C— CH2— CH— COOH 

^      \ 
\^CH 

NH 

(tryptophane) 

readily  yields,  through  splitting  off  the  NH2  group  and  addition  of    H,  indole 
propionic  acid  (formerly  incorrectly  called  skatole  acetic  acid),  which  is 

CH2— CH2— COOH 

CH 

1       / 
NH 

and  from  which  in  turn  may  rcadilj'  be  formed  indole  acetic  acid  (erroneously  called 
skatole  carboxylic  acid),  which  is 

CH2— COOH 


\     /' 


NH 


\ 
CH 

/ 


580 


GASTRO-INTESTINAL  "AUTOINTOXICATION " 


Both  of  these  substances  have  been  found  in  the  intestinal  contents.     From  them 
are  formed  the  better  known  skatole, 


CH., 


and  indole, 


NH 

In  dogs,  but  not  in  man,  kynurenic  acid. 


is  also  formed  from  tryptophane.^' 

The  greatest  interest  concerning  these  bodies  arises  from  the  fact  that  after 
they  are  absorbed  from  the  intestine  they  become  combined  with  sulphuric  or 
glycuronic  acid,  and  are  excreted  in  the  urine  as  salts,  of  these  acids;  consequently 
the  amount  of  sulphuric  acid  appearing  in  the  urine  in  such  organic  combination 
(■'ethereal  sulphuric  acid")  is  considered  as  an  index  of  the  amount  of  intestinal 
putrefaction.  In  the  case  of  indole  and  skatole,  which  have  no  hydroxyl  group, 
a  preliminary  oxidation  occurs,  whereby  indole  is  converted  into  indoxyl, 


and  skatole  into  skatoxyl,  

/     \— C— CH3 
^-{        \ 

\  COH 

\       / 
NH 

and  they  are  then  combined  with  sulphuric  or  glycuronic  acid,  as  follows; 


<' 


-C— 
CH 


OH  +H 


O-SO2-OK 


\     /" 


-C— O— SO2— OK 
\ 
CH       (indican) 


HN 


NH 


By  far  the  greater  part  of  these  aromatic  substances,  when  excreted 
in  the  urine,  is  combined  with  sulphuric  acid,  and  but  a  small  part 
with  glycuronic  acid;-^  but  in  case  the  amount  of  sulphuric  acid  avnil- 


"  See  Elliiigor,  Zeit.  physiol.  Chem.,  1901  (13),  325. 

=*  Shorwiu  (.lour.  Hiol.  Chem.,  1917  (31),  307)  states  that  phenylacetic  acid  is 
excreted  by  monkeys  combined  with  glycine,  but  by  num  it  is  excreted  combined 
with  glutamine. 


INDOLE  AND  INDICAN  581 

able  is  too  siiiull  to  coiubine  with  all  the  aromatic  railicals  cntcrinK  the 
blood,  a  large  amount  of  the  glycuronic  acid  compound  appears  in  the 
urine  {e.  g.,  after  therapeutic  administration  of  phenol,  cre.sol,  thymol, 
(iamphor,  etc.)-  Both  the  preliminar\'  oxidation  and  the  cond:)ininK 
with  acids  seem  to  occur  chieHy  in  the  liver,  this  process  constitutinjj; 
one  of  the  most  important  of  the  many  protective  offites  of  that  organ, 
since  the  resulting  compounds  are  much  less  toxic  than  are  the  original 
substances. ^^  Herter  and  '\Vakeman^"have  shown  that  living  cells  have 
the  power  of  acting  upon  indole  and  phenol  (and  presumably  upon  the 
rest  of  this  group)  in  such  a  way  that  they  cannot  be  recovered  by  dis- 
tillation. Most  active  in  this  respect  is  the  liver,  then  in  order  come 
kidney,  muscle,  blood,  and  brain.  The  change  seems  to  be  a  loose 
chemical  combination  with  the  protoplasm  of  the  cells,  and  the  power 
of  the  tissues  to  bring  about  this  combination  is  not  greatly  decreased 
by  serious  pathological  changes  in  the  organs  (e.  g.,  ricin  poisoning). ^^ 
Indole. — This  is  probably  the  most  important  member  of  this 
group  of  substances,  the  striking  color  of  its  derivatives  making  its 
detection  in  the  urine  easy,  so  that  it  is  generally  used  as  the  most 
available  index  of  the  amount  of  putrefaction  that  is  occurring  in  the 
intestines. ^^  The  greatest  quantities  are  found  when  intestinal  putre- 
faction is  marked,  especially  in  intestinal  obstruction  involving  the 
small  intestine;  obstruction  of  the  large  intestine,  as  Jaffe  first  demon- 
strated, does  not  cause  marked  indicanuria  unless  the  stagnation 
involves  the  ileum,  as  it  may  in  the  later  stages  of  obstruction.  With 
marked  impairment  of  renal  function  indican  may  accumulate  in  the 
blood  (see  Uremia).  There  can  be  no  question  that  the  indican  of  the 
urine  is  derived,  at  least  in  part,  from  the  indole  formed  in  the  intes- 
tine, for  administration  of  indole  by  mouth  to  either  animals  or  man 
causes  a  considerable  increase  in  the  indican  present  in  the  urine; 
however,  but  40  to  60  per  cent,  can  be  recovered  in  this  way,  the  rest 
apparently  being  oxidized  to  other  compounds,  part  of  which  may  also 
appear  in  the  urine. ^^  Whether  part  of  the  urinarj^  indican  is  derived 
from  tryptophane  liberated  during  intracellular  protein  metabolism, 
and  not  from  intestinal  putrefaction,  has  long  been  a  disputed 
point  among  physiological  chemists.^"*  The  demonstration  by  Ellinger 
and  Gentzen^^  that  tr3^p':ophane,  when  fed  or  injected  subcutaneously, 
causes  no  increase  in  urinary  indican,  whereas  its  injection  into  the 
cecum  causes  much  indicanuria,  w-ould  indicate  that  indole  is  formed 

-^  Metchnikoff  insisted  that  these  sulfo-compounds  still  retain  considerable 
toxicity.      (Ann.  Inst.  Pasteur,  1914  (27),  S93). 

3"  Jour.  Exper.  Med.,  1899  (4),  307. 

"  For  further  discussion  of  this  topic,  see  "Chemical  Defences  against  Poisons 
of  Known  Composition,"  Chapter  x. 

32  See  Houghton,  Amer.  Jour.  Med.  Sci..  190S  (135),  567. 

3'  If  gelatin  is  substituted  for  proteins  in  the  dietary,  indican  is  not  excreted, 
because  gelatin  does  not  contain  tryptophane  (Underbill,  Amer.  Jour.  Phvsiol., 
1904  (12),  176). 

'*  Literature  bv  Gerhardt,  Ergeb.  der  Phvsiol..  1904  (III,  Abt.  I),  131. 

"  Hofmeister's  Beitr.,  1903  (4),  171. 


582  G ASTRO-INTESTINAL  "AUTOINTOXICATION" 

from  tryptophane  only  through  putrefaction,  and  not  in  cellular  me- 
tabolism. Other  experiments  support  the  same  view.^^  However, 
it  is  possible  that  part  of  the  indican  present  in  the  urine  during  con- 
ditions associated  with  gangrene,  putrid  cancers,  putrid  placentas, 
or  putrid  purulent  exudates,  may  be  derived  from  these  decomposing 
materials.  The  statement  that  indicanuria  is  of  significance  in  in- 
sanity could  not  be  substantiated  by  Borden, ^^  who  used  quantitative 
methods  and  careful  controls.  A  large  proportion  of  the  data  and 
conclusions  in  the  literature  concerning  indicanuria  are  valueless  be- 
cause of  improper  or  inadequate  methods. 

Probably  the  chief  agent  in  the  formation  of  indole  in  the  intes- 
tines and  in  putrid  tissues  is  the  colon  bacillus,  which,  as  is  well  known, 
produces  indole  in  ordinary  culture-media.^^ 

Toxicity  of  Indole. — Although  the  toxicity  of  indole  seems  to  be 
relatively  slight,  and  this  toxicity  is  further  reduced  by  the  conver- 
sion of  indole  into  indoxyl  and  indican,  yet  Hei'ter^^  found  that  ad- 
ministration to  healthy  men  of  indole  in  quantities  of  0.025  to  2  grams 
per  day  caused  frontal  headache,  irritability,  insomnia,  and  confusion; 
the  continued  absorption  of  enough  indole  to  cause  a  constant  strong 
reaction  for  indican  in  the  urine  is  sufficient  to  cause  neurasthenic 
symptoms.  Long-continued  injection  of  indole  leads  to  hypertrophy 
of  the  adrenal  medulla  and  slight  interstitial  changes  in  the  kidneys,"^" 
but  the  reputed  responsibility  of  indole  for  arteriosclerosis  is  most 
doubtful. ^^  Lee*^  has  also  demonstrated  that  indole,  skatole,  and 
methyl  mercaptan  cause  muscles  to  react  to  stimuli  like  fatigued 
muscles.  Normal  urine  contains  but  about  12  milligrams  of  indican 
per  day,  which  amount  is  so  insignificant  in  proportion  to  the  above- 
mentioned  doses  that  were  found  necessary  to  produce  symptoms, 
that  we  may  well  doubt  the  occurrence  of  noticeable  intoxication 
from  this  substance  under  ordinary  conditions.  Nesbitt*^  states  that 
twenty  times  as  much  indole  or  skatole  as  are  excreted  daily  by  an 
adult  man  may  be  injected  into  the  jugular  vein  of  a  dog  of  four 
kilos  without  causing  appreciable  effects.  Richards  and  Howland, 
however,  have  demonstrated  the  possibility,  that  defective  oxidation 
of  substances  of  this  group  may  permit  of  intoxication. ■'■'  When 
subcutaneously  injected,  dissolved  in  oil,  indole  and  skatole  have  the 
property  of  greatly  stimulating  epithelial  proliferation,  similar  to  the 

'«  See  Scholz,  Zeit.  physiol.  Chein.,  1903  (38),  513;  Undcrhill,  loc.  cil.  Sherwin 
and  Hawk  found  an  absence  of  indican  in  the  urine  in  the  latter  part  of  a  long 
fast  (Biochem.  Bull.,  1914  (3),  41G). 

"  Jour.  Biol.  Cheiii.,  1907  (2),  575. 

'*  See  Distaso  and  Sugden,  Biochem.  Jour.,  1919  (13),  153. 

'»  New  York  Med.  Jour.,  1898  (l58),  89. 

"  Woolley  and  Newhurgh,  Jour.  Amor.  Med.  Assoc,  1911  (50),  1790. 

^1  See  Steenhuis,  Folia  Mikrohiol.,  1915  (3),  70. 

"Jour.  Amer.  Med.  Assoc,  1900  (-40),  1499. 

^'  Jour.  E.xper.  Med.,  1899  (4),  5. 

*"  See  note  in  Science,  1906  (24),  979. 


PRODUCTS  OF  I'l'TREFACTION  583 

action  of  scarlet  U  and  other  fat  stains  (Stoebcr),'*''  but  we  have  no 
direct  evidence  that  these  substances  cause  similar  effects  in  the  human 
body. 

Other  Aromatic  Compounds. — Skatole  seems  to  accompany  indole  in  small 
amounts,  Init  api)arently  in  no  constant  (luantitative  relation.  Herter"  states 
that  skatole  is  formed  under  entirely  dilTerent  conditions  from  indole,  and  that 
B.  call  does  not  i)roduce  skatole.  It  is  not  always  present  in  the  contents  of  the 
large  intestines  of  healthy  persons,  and  seems  to  be  formed  later  than  indole. 

Indole-acetic  acid  appears  in  the  normal  urine  in  extremely  mimite  cpiantities, 
and  is  increased  in  tlie  same  conditions  as  skatole.  It  is  the  mother  substance  of 
urowscin,  and  can  be  found  in  the  intestines  of  patients  who  show  this  substance  in 
the  urine  (Herter).''"  Ross^*  found  indole-acetic  acid  in  the  urine  in  21  i)er  cent, 
of  normal  persons,  and  in  48  per  cent,  of  dementia  precox  cases,  and  obtained  evi- 
dence in  favor  of  an  endogenous  origin  in  two  cases  studied  especially  to  determine 
this  point. 

Fheuylacelic  acid*^  is  formed  from  phenylalanine  on  putrefaction,  as  also  are 
phenylpropionic  and  benzoic  acids.  Benzoic  acid  combines  with  glycine  and  is 
excreted  as  hii)p\iric  acid,  and  the  phenylpropionic  acid  is  excreted  as  />-hydroxy- 
hippuric  acid.  Phenylacetic  acid  combines  with  glutamine  and  is  excreted  as 
phenylacetylglutamine.  It  is  but  slightly  toxic,  5  gm.  doses  in  man  causing  slight 
symptoms  resembling  alcoholic  intoxication;  10  gms.  not  producing  serious  results. 

PhenoP"  appears  in  the  urine  normally  in  very  minute  quantities — from  0.005 
to  0.07  grams  per  day,  according  to  various  observers.  These  figures  are  undoub- 
tedly too  low,  for  Folin  and  Denis^'  found  the  total  excretion  of  phenols  to  be 
from  0.2  to  0.4  gm.  per  day,  the  amount  varying  with  the  protein  intake."-  They 
seem  to  come  chiefly,  if  not  entirely,  from  tyrosine.*'  Much  more  is  undoubtedly 
formed  in  the  intestines,  for  but  a  small  fraction  of  phenol  given  by  mouth  (2 
to  3  per  cent.,  according  to  Munk)  appears  in  the  urine  as  a  sulphuric-acid  com- 
pound; part  of  the  rest  is  oxidized  to  hydrochinon  and  pyrocatechin,  C6H4(OH)2, 
and  eliminated  as  ethereal  sulphates.  These  sulphates,  although  distinctly  toxic 
are  much  less  so  than  the  phenol  itself  (Metchnikoff).*^  Contrary  to  prevailing 
ideas,  Folin  and  Denis  found  the  greater  part  of  the  phenols  to  be  excreted  uncom- 
bined.  The  largest  quantities  are  found  in  the  same  conditions  as  indican  ex- 
cept, of  course,  in  "carbolic-acid"  poisoning,  when  the  amounts  may  be  so  great 
that  practically  all  the  sulphuric  acid  in  the  urine  is  in  this  organic  combination, 
much  of  the  phenol  under  these  conditions  being  also  combined  with  glycuronic 
acid.*^  This  pairing  is  accomplished  chiefly  in  the  liver  (Dubin).  A  small  amount 
of  urinary  phenol  may  be  of  endogenous  origin. ^^  Rhein*®  ascribes  the  phenol  of 
intestinal  origin  to  the  action  of  a  specific  sort  of  colon  bacilli,  B.  coli  phenologenes, 
which  does  not  produce  indole  but  produces  phenol  from  tyrosine,  and  also  from 
p-hydroxybenzoic  acid  which  therefore  may  be  an  intermediary  in  tyrosine  cleav- 

Blood  contains  small  amounts  of  free  phenols,  about  one-third  being  poly- 
phenols, none  being  conjugated.  In  a  series  of  pathological  cases  Theis  and  Bene- 
dict" found  from  1.87  to  7.96  mg.  per  100  cc.  blood,  somewhat  higher  figures  being 
found  in  sarcoma  and  hernia  cases  than  in  other  diseases. 

Cresol  (chiefly  paracresol),  para-oxy phenyl  acetic  acid,  and  para-oxy phenyl  pro- 

«  Mtinch.  med.  Woch.,  19,10  (57),  947. 
■"^  Jour.  Biol.  Chem.,  1908  (4),  101;  general  discussion. 
''  Jour.  Biol.  Chem.,  1908  (4),  253. 
^8  Arch.  Int.  Med.,  1913  (12),  112  and  231. 
^^  See  Sherwin  and  Kennard,  Jour.  Biol.  Chem.,  1919  (40),  259. 
^°  Literature  given  by  Dubin,  Jour.  Biol.  Chem.,  1916  (26),  69. 
"  Jour.  Biol.  Chem.,^  1915  (22),  309. 
"  Moore,  Amer.  .Jour.  Dis.  Child.,  1917  (13),  15. 
"  Tsudji,  Jour.  Biol.  Chem.,  1919  (38),  13. 
"  Ann.  Inst.  Pasteur,  1910  (24),  755. 

"  See  the  observations  of  Wohlgemuth  and  of  Blumenthal  (Biochem.  Zeit- 
schrift,  1906  (1\  134),  on  the  detoxication  of  lysol  and  similar  poisons. 
56  Biochem.  Zeit.,  1917  (84),  246. 
"  Jour.  Biol.  Chem.,  1918  (36),  99. 


584  G ASTRO-INTESTINAL  "AUTOINTOXICATION" 

pionic  acid  appear  under  similar  conditions,  except  that  the  two  oxy-acids  are 
possibly  also  formed  within  the  body  through  cellular  metabolism,  as  they  have 
been  found  present  in  the  urine  independent  of  intestinal  putrefaction.  Para- 
cresol  is  quantitatively  the  most  important  of  the  urinary  phenols,  and  long  con- 
tinued feeding  produces  no  noticeable  effects  in  rabbits.''*  Probably  part  of  the 
benzoic  acid  that  appears  in  the  urine  combined  with  glycine,  as  hippuric  acid,  is 
derived  from  intestinal  putrefaction.*^ 

The  Pressor  Bases^" 

Among  the  products  of  protein  putrefaction  are  several  amines 
that  have  marked  power  to  stimulate  the  sympathetic  nervous  system 
and  thus  to  raise  the  blood  pressure,  hence  resembling  epinephrine 
physiologically  as  well  as  chemically.  There  are  also  bases  derived 
from  amino-acids  which  stimulate  non-striated  muscle  without  raising 
general   blood   pressure.     The  most  important   of  these    bases   are: 

Phemjl-ethylamine  C6H5.CH2.CH0.NH2, 

derived  from  phenylalanine  C6H6.CH2CHNH2.COOH. 

Para-hydroxy-phenyl-ethylamine  (tyramine)  OH.C6H4.CH>.CH-..XH2 

derived  from  tyrosine.  OH.C6H4.CH2.CHNH2.CbOH. 

Its  relation  to  epinephrine  is  seen  on  comparing  with  the  structural  formula  of 
the  latter  (OH)2C6H3.CHOH.CH2NH.CH3 

/NH— CH 
Beta-iminazolyl-ethylarnine  {histamine)  CH/^  : 

^N C— CHoiCHo.NH. 

.NH— CH 
derived  from  histidine  CH/' 

^N C— CH2.CHNH2.COOH 

It  will  be  observed  that  these  are  all  amines  derived  from  the 
cyclic  amino-acids  of  proteins  by  the  process  of  decarhoxylization 
(loss  of  CO2).  The  straight  chain  amines  are  much  less  active 
physiologically.  The  lowest  amine  having  any  considerable  pressor 
action  is  isohutylamine,  but  para'phenylamine  is  at  least  five  times  as 
active  as  any  aliphatic  amine  (Barger).  Tyramine  injected  subcutan- 
eously  or  intravenously  increases  blood  pressure  and  slows  the  pulse 
rate,''^  resembling  epinephrine,  but  it  is  onl}^  one-twentieth  as  active. 
Histamine  causes  ordinarily  a  fall  of  blood  pressure,  although  it  does 
constrict  many  peripheral  vessels.  Capillaries  are  dilated  by  his- 
tamine and  Dale  and  Laidlaw  compare  it  to  a  capillary  i)oison.''^°  Its 
most  striking  effect  is  in  causing  profound  contraction  of  the  uterine 
and  bronchial  muscle.  The  relative  effects  of  epinephrine,  tryamine 
and  histamine  are  given  by  Barbour,  as  follows : 

*s  Denny  and  Frothingham,  Jour.  Med.  Res.,  1914  (31),  277. 

"  See  Prager,  Med.  News,  1905  (86),  1025;  Magnus-Levy,  Munch  med.  Woch., 
1905  (52),  21G8. 

*"  Full  bibliography  given  by  Barger,  "The  Simpler  Natural  Bases, "^Mono- 
graphs on  Biochemistry,  London,  1914. 

•iSee  Hewlett,  Proc.  Soc.  Exp.  Biol.  Med.,  1917  (15),  12. 

""See  also  Dale  and  Richards,  Jour.  Physiol.,  1918  (52),  110. 


THE  PRESSOR  BASES 


585 


Epinephrine 

Tyrainine   (p-hydroxyphenyl 
ethylamine) 

Histamine          (/3-iminazolyl- 
ethylannine) 


Blood 
pressure 

Peripheral 
vessels 

Coronary 
vessels  (Ox) 

\on-prr>({nant 
uterus 

+ 

+ 

- 

- 

+ 

+ 

+ 

- 

_ 

+ 

+ 

+ 

+  means  rise  of  blood  pressure  or  constriction, 
amine  may  have  a  pressor  effect  in  some  animals. 


the  opposite;  the  last-named 


Another  difference  is  the  production  of  severe  urticarial  reactions  by 
histamine  introduced  into  the  skin,  while  tyramine  and  epinephrine 
both  cause  local  blanching  (Sollnian  and  Pilcher).''- 

Thcse  substances  have  all  been  found  in  putrid  protein  materials, 
produced  by  the  action  of  anaerobic  bacteria,  and  possibly  they  are 
formed  in  the  intestines,  as  colon  bacilli  are  able  to  form  histamine 
from  histidine.*'^  Para-hydrox3'-phenyl-ethylaniine  is  also  one  of  the 
active  constituents  of  ergot,  and  has  been  found  in  the  salivary  gland 
of  cephalopods,  where  it  functions  as  a  venom  in  paralyzing  the  prey. 
Beta-iminazolyl-ethylamine  is  said  to  be  the  most  important  con- 
stituent of  ergot.  It  has  been  found  regularl}^  present  in  the  intestinal 
mucosa,  presumably  formed  by  intestinal  bacteria.  According  to 
AbeP^  histamine  is  widely  distributed  in  all  animal  tissues,  organ 
extracts,  Witte's  peptone,  etc.,  or  at  least  some  substance  that  has  simi- 
lar physiological  effects.  He  believes  it  to  be  especially  abundant  in 
the  hypophysis  and  to  form  its  chief  active  constituent,  but  histamine 
exhibits  distinct  differences  from  pituitrin.  Hanke  and  Koossler^^" 
however,  have  shown,  by  using  purely  chemical  methods,  that  the 
perfectly  fresh  hypophysis  contains  no  histamine.  They  could  further 
demonstrate  that  peptone  prepared  from  fibrin  under  aseptic  condi- 
tions is  free  from  histamine  yet  capable  of  producing  typical  peptone 
shock. 

All  these  amines  are  detoxicated  by  the  liver,  and  hence  have  little 
effect  when  given  by  mouth. ^^  It  is  probable  that  no  inconsiderable 
amounts  are  taken  in  our  food  and  formed  in  the  intestines  every  day 
(Abel).  Their  detoxication  is  accomplished  by  deaminization  and 
oxidation,  the  resulting  carboxylic  acids  being  excreted  or  burned. 
Therefore  it  is  not  certain  whether  pressor  bases  formed  in  the  intes- 
tines ever  have  any  pathological  effect,  but  it  is  quite  possible  that 
outside  the  portal  territory  various  infections  maj^  give  rise  to  pressor 

"  Jour.  Pharm.,  1917  (9),  391. 

63  Koessler  and  Hanke,  Jour.  Biol.  Chem.,  1919  (39),  539. 

"  Jour.  Pharmacol.,  1919  (13),  243. 

«^»Jour.  Biol.  Chem.,  1920. 

6"  See  Guggenheim  and  LoefBer,  Biochem.  Zeit.,  1916  (72;,  325. 


586  GASTRO-INTESTINAL  "AUTOINTOXICATION'' 

bases  which  enter  the  general  circulation  directly  and  escape  the  de- 
fensive mechanism  of  the  liver.  They  may  also  cause  local  effects 
in  the  tissues  where  they  are  formed,  e.  g.,  the  bronchi.^"" 

In  some  respects  their  effects  resemble  those  of  anaphylactic 
intoxication,  and  as  the  latter  apparently  results  from  toxic  products 
of  protein  cleavage  the  possibility  that  here  too  pressor  bases  are  con- 
cerned at  once  presents  itself,  but  as  yet  the  relation  is  undetermined 
(see  Anaphylaxis,  Chap.  viii).  The  resemblance  is  especially  seen  in 
the  profound  effect  on  the  bronchial  musculature,  which  can  be  thrown 
into  strong  contraction,  especially  by  beta-iminazoljdethylamine 
which  in  0.5  mg.  doses  kills  guinea  pigs  from  asphyxia,  with  distended 
lungs  as  in  fatal  anaphylaxis ;  also  it  causes  a  similar  fall  in  temperature. 
Another  point  of  similarity  is  the  severe  local  urticaria  when  weak 
solutions  (1-1000)  of  histamine  are  placed  on  a  scarified  area  of  skin,^^ 
recalling  vividly  the  fact  that  urticarial  eruptions  are  conspicuous  in 
some  types  of  anaphylactic  reactions.  Furthermore,  in  guinea  pigs 
histamine  kills  by  bronchial  spasm,  in  rabbits  by  obstruction  to  the 
pulmonary  circulation  (Dale  and  Laidlaw),"  which  difference  is  also 
characteristic  of  anaphylaxis  in  these  animals.  On  the  other  hand, 
histamine  does  not  produce  the  profound  alteration  in  the  coagulability 
of  the  blood  that  is  characteristic  of  anaphylaxis  and  of  peptone  shock, 
but  it  may  be  that  in  each  of  these  cases  some  other  poison  is  respon- 
sible for  the  effect  on  the  blood,  in  addition  to  histamine  or  a  similar 
substance.  In  doses  of  1  mg.  and  upward  in  cats,  histamine  causes  a 
condition  resembling  traumatic  shock,  there  being  oligsemia  from 
passage  of  plasma  out  of  the  vessels  and  retardation  of  blood  in  the 
periphery  because  of  loss  of  tone  by  the  capillaries.  Possibl}^  in  trau- 
matic shock  histamine  is  liberated  in  the  injured  tissues. 

Alkaptonuria's 

Alkaptonuria  may  be  appropriately  considered  in  this  connection, 
since  it  depends  on  an  abnormal  metabolism  of  the  aromatic  groups, 
tyrosine  and  phenylalanine,  which  are,  partly  at  least,  split  out  of 
the  protein  molecule  in  the  intestine.  This  condition  is  characterized 
by  the  tendency  of  the  urine  to  turn  dark  on  exposure  to  air,  due  to  the 
presence  in  it  of  homogentisic  acid.^^     Homogentisic  acid   has  been. 

"^"Compare  K.  K.  Koessler,  "The  pathogenesis  of  bronchial  asthma,"  Arch 
Int.  Med.,  1920.  In  this  article  asthma  is  considered  as  an  aminosis  (amine 
intoxication). 

"  Eppinp;er  and  Guttmann,  Zeit.  klin.  Med.,  1913  (78),  399. 

«'  Jour.  Physiol.,  1919  (.52),  355. 

08  R6suin6  and  literature  by  Falta,  Biochem.  CeiitrallUatt,  1904  (3),  174,  and 
Deut.  Arch.  klin.  Med.,  1904  (81),  231;  Garrod,  "Inborn  Errors  of  Metabolism," 
Oxford  Med.  Publications,  1909;  also  Lancet,  July,  1908;  Fronunlunz,  Biochem. 
Centr.,  190S  (S),  1. 

*"  It  shouUl  be  inentioned  that  hydrochinon,  when  present  in  the  urine  (usually 
after  infi;estion  of  large  quantities  of  phenol),  may  also  turn  dark  on  exiK)sure 
to  air;  and  melanin,  may  l)e  excreted  as  a  clironiof^en  which  turns  dark  on  ex- 
posure', l)y  patients  with  melanotic  tumors  or  ochronosis  (</.  t\). 


ALKAPTONURIA  587 

found  in  the  blood  but  not  in  the  feces  of  alkaptonurics,  and  the  urine 
shows  no  other  deviations  from  the  normal  except  a  slight  increase  in 
ammonia,  with  which  the  acid  is  combined.  It  is  of  ran;  ocj'urrence. 
persists  throughout  life  with  but  little  apparent  effect  upon  the  health 
of  the  individual,  and  is  often  hereditary,  being  grouped  by  Clarrod 
along  with  cystinuria,  pentosuria  and  albinism  as  a  "chemical  malfor- 
mation "  of  hereditary  origin.^"  The  relation  of  these  aromatic  bodies 
to  the  aromatic  constituents  of  the  proteins  is  best  shown  by  comparing 
their  structural  formulae : 

Phenylalanine,  /     NcH,— CHNHj— COOH. 

Tyrosine,  HO^     \CH2— CHNH,— COOH. 

_0H 
Uroleucic  acid,^i  /     NcHa— CHOH— COOH. 

H0~ 
_0H 
Homogentisic  acid,      <^      y  CH-. — COOH. 
H0~ 

Apparently  the  condition  depends  upon  an  abnormality  in  the  inter- 
mediary metabolism,  and  not  upon  an  abnormal  formation  of  homo- 
gentisic acid  through  intestinal  putrefaction,  as  was  at  first  believed. 
Alkaptonuria  is  never  observed  in  slight  degrees;  if  there  is  any 
homogentisic  acid  in  the  urine  at  all  it  is  there  in  large  amounts  (4-5 
grams  per  day),  depending  on  the  diet,  for  when  the  error  in  metabo- 
hsm  is  present  at  all  it  is  complete.  On  a  mixed  diet  the  ratio  of 
homogentisic  acid  to  nitrogen  in  the  urine  is  40-50  to  100.  The  pre- 
vailing idea  has  been  that  the  abnormality  consists  not  in  the  excessive 
formation  of  homogentisic  acid,  but  in  a  lack  of  ability  on  the  part 
of  the  alkaptonuric  individual  to  split  open  the  benzene  ring.  It  is 
generally  stated  that  tyrosine  and  phenylalanine  first  suffer  a  splitting 
out  of  the  nitrogen  radical  from  the  alanine  side-chain,  and  then  are 
oxidized  into  homogentisic  acid,  following  which  changes  comes  a 
disintegration  of  the  benzene  ring,  with  subsequent  complete  oxidation. 
On  this  basis  the  alkaptonuric  accomplishes  the  conversion  into  the 
oxj'-acid  but  the  process  stops  there.  Wakeman  and  Dakin,"'-  how- 
ever, have  obtained  evidence  that  in  the  normal  oxidation  of  tyrosine 

~°  Alkaptonurics  may  give  a  positive  Wassermann  reaction  without  other  evi- 
dence of  syphilis,  and  in  one  case  this  reaction  disappeared  when  the  patient 
was  given  large  amounts  of  tvrosine  (Soderbergh,  Nord.  Med.  Arkiv.,  1915  (48), 
1). 

^^  The  older  writers  stated  that  uroleucic  acid  commonly  accompanied  homo- 
gentisic acid  in  the  urine  of  alkaptonuria,  but  later  observations  do  not  conhrm 
this.     (Oswald,  Zeit.  phvsiol.  Chem.,  1914  (93),  307). 

■-  Jour.  Biol.  Chem.,  1911  (9),  139  and  151. 


588  GASTRO-INTESTINAL  "AUTOINTOXICATION" 

and  phenylalanine,  homogentisic  acid  is  not  an  intermediary  product, 
and  Dakin  states  that  the  alkaptonuric  can  destroy  simple  derivatives 
of  phenylalanine  and  tyrosine,  provided  their  structure  is  such  that  the 
formation  of  substances  of  the  type  of  homogentisic  acid  is  precluded 
He  believes  that  in  alkaptonuria  there  is  abnormal  formation  of 
homogentisic  acid  as  well  as  a  failure  to  destroy  it  when  formed.  On 
the  other  hand,  Abderhalden^^  has  been  able  to  cause  the  appearance 
in  the  urine  of  homogentisic  acid  in  a  normal  individual  by  feeding 
large  amounts  of  tyrosine,  which  is  in  favor  of  the  view  that  it  is  a  normal 
intermediary  in  tyrosine  catabolism.'^'*  In  any  case  the  administration 
of  tyrosine  or  phenylalanine,  or  of  tyrosine-rich  foods — i.  e.,  proteins — 
causes  a  marked  increase  in  the  amount  of  homogentisic  acid  eliminated 
in  the  urine;  indeed,  this  increase  is  almost  quantitative.  Normal 
individuals  when  given  these  substances  in  moderate  amounts,  or 
homogentisic  acid  itself,  destroy  them  completely,  so  that  the  latter 
does  not  appear  at  all  in  the  urine. ''^  If  alkaptonurics  are  kept  with- 
out protein  food  for  some  time,  the  elimination  of  alkaptonuric 
acid  goes  on,  although  in  diminished  amounts,  indicating  that  the 
aromatic  amino-acids  formed  in  tissue  catabolism  also  fail  to  be  de- 
stroyed and,  therefore,  appear  in  the  urine  as  these  derivatives. 
Since  gentisic  acid, 

OH 
/^— COOH, 
HO 

when  given  by  mouth,  is  also  eliminated  unchanged  by  alkaptonurics, 
although  completely  destroyed  by  normal  individuals,  it  seems  evident 
that  the  difficulty  in  metabolism  affects  the  benzene  ring  itself  and 
does  not  depend  upon  the  character  of  the  side-chain.  Normal  organ- 
isms seem  to  be  capable  of  destroying  such  aromatic  compounds  as 
pass  through  a  stage  of  homogentisic  acid  in  being  oxidized,  which 
indicates  that  the  benzene  ring  can  be  broken  up  only  when  oxidized 
in  this  particular  manner  (the  2,  5  position) ;  the  alkaptonuric  differs 
in  being  unable  to  break  up  even  this  form  (Falta).  According  to 
Garrod'^'^  the  conversion  of  tyrosine  and  phenjdalanine  into  homogen- 
tisic acid  is  so  complete  that  the  ratio  of  homogentisic  acid  to  nitro- 
gen is  constant  and  the  same  in  all  cases.  Frommhcrz  and  Her- 
manns'^ advance  the  suggestion  that  normal  oxidation  of  tlic  aromatic 
radicals  may  take  place  by  two  routes,  one  b}'  way  of  homogentisic 
acid,  the  other  by  way  of  the  3-4  dioxy-derivatives  (i.  e.,  pyrocate- 
chin),  since  such  derivatives  can  be  readilj^  oxidized  in  the  metabolism 

'3  Zeit.  physiol.  Chem.,  1912  (77),  454. 

'\Katsch  (Deut.  Arch.  klin.  Med.,  1918  (127),  210)  believes  that  homogentisic 
acid  is  converted  into  acetone. 

'^  Gross  states  that  normal  serum  destroys  homogentisic  acid,  which  property 
is  lacking  in  the  serum  of  alkai)tonurics  (Hiochem.  Zeit.,  1914  (til),  1C5). 

"  Garrod  and  Clarke,  Biochem.  Zeit.,  1907  (2),  217. 

"  Zeit.  physiol.  Chem.,  1914  (91),  194. 


POISONOUS  AMINES  589 

of  alkaptonurics  who  cannot  destroy  lioniogentisic  acid.  That  is, 
their  deficiency  involves  only  one  of  two  possible  methods  of  oxidizing 
aromatic  compounds,  leaving  them  considerable  capacity  for  this  im- 
portant metabolic  function.  The  tissues  of  the  alka[)tonuric  are  prob- 
ably not  chemically  affected  in  this  condition,  for  Abderhalden"  found 
that  the  hair  and  nails  of  an  alkaptonuric  contained  normal  propor- 
tions of  tyrosine.  There  is  often  an  arthritis,  from  deposition  of  pig- 
ment in  the  joints,  designated  ))y  Gross^^  as  "arthritis  alkaptonurica." 
In  some  cases  of  alkaptonuria  a  pigmentation  of  the  cartilages  also 
occurs,  ochronosis,  but  the  association  is  not  constant;  ochronosis  may 
occur   without   alkaptonuria,   and   conversely.     (See  "Ochronosis.") 

(2)  Substances  Arising  from  the  Fatty  Acid  Radicals  (Amino  Acids) 

OF  Proteins 

As  stated  in  the  introductory  chapter,  the  protein  molecule  consists  of  a  com- 
bination of  a  great  number  of  organic  acids,  of  various  sorts,  all  of  which  have  as  a 
common  characteristic  the  presence  of  a  NH-.  group  attached  to  the  carbon  atom 
nearest  the  acid  radical,  the  a  position;  thus,  R  — CHXHo  — COOH.  A  few  of 
the  amino  acids  contain  an  aromatic  group,  and  the  relation  of  these  to  intestinal 
decomposition  has  been  considered  above.  The  greater  number  have  a  simple 
fatty  acid  radical  (the  simplest  amino-acid  being  glycine,  CHoXHj  — COOH),  and 
from  them  are  derived  by  intestinal  putrefaction  substances  that  are,  for  the  most 
part,  chemically  simple  and,  as  far  as  known,  pathologically  unimportant.  From 
leucine  alone  is  derived  a  substance  of  known  coasiderable  toxicitv,  the  pressor 
CH. 

base  isoatnylamine.  ^  CH— CH2  — CH2  — XH-j  which  is  less  powerful  than 

CH3 
the  cyclic  pressor  bases  described  previously.     Bain'^"  found  it  the  most  abundant 
pressor  base  of  the  urine. 

Fatty  acids  may  readily  be  formed  from  them  by  splitting  out  of  the  NHj 
group;  thus  acetic  acid  may  be  formed  from  glycine,  propionic  acid  from  alanine, 
etc.  Apparently  butyric  and  acetic  acid  are  the  acids  most  commonly  formed  in 
this  way.  Gaseous  derivatives,  such  as  hydrogen,  ammonia,  carbon  dioxide,  and 
marsh-gas,  are  also  produced.  Acetone  is  perhaps  formed  from  these  fatty  acids; 
it  is  often  present  in  the  intestinal  contents,  but  may  come  from  other  sources. 

Certain  conditions  of  cyanosis  have  been  designated  as  enterogenous  cyanosis, 
(See  Methemoglobin,  Chap,  xviii)  because  of  the  belief  that  the  methemoglobin 
responsible  for  the  cyanosis  is  caused  by  nitrites  derived  from  intestinal  putrefac- 
tion and  demonstrable  in  the  blood.*"  Presumably  the  nitrites  come  from  the 
XHo  groups  of  the  protein  molecule,  the  colon  bacillus  being  an  active  former  of 
nitrites.  Under  the  same  term  are  included  the  cases  of  snlph-hemoglobinemia. 
This  condition  is  ascribed  by  Wallis*'  to  bacteria  which  produce  from  the  proteins 
a  hydroxylamine  derivative,  capable  of  reducing  oxyhemoglobin,  and  which  he 
finds  present  in  the  blood  of  patients  with  sulph-hemoglobinemia. 

Diamines. — Of  much  interest  are  the  substances  that  are  formed  from  the 
amino-acids  by  bacterial  action,  which  still  retain. their  nitrogen  radicals — the 
ptomains  (See  Chap.  IV).  Two  of  these,  the  diamines  putrescine,  XHo  (CHs)^  XH;, 
and  cadaverine,  XH2(CH2)5  NH2  are  of  particular  interest,*'-  because  they  have  been 

'8  Zeit.  phvsiol.  Chem.,  1907  (52),  435. 

"  Deut.  Arch.  klin.  Med.,  1919  (128)  249. 

'9'^  Quart.  Jour.  Exp.  Phvsiol.,  1914  (8).  229. 

»«See  Gibson,  Quart.  Jour.  Med.,  1907  (1),  29;  West  and  Clarke,  Lancet, 
Feb.  2,  1907;  Davis,  Lancet,  Oct.  26,  1912. 

"  Quart.  Jour.  Med.,  Oct.,  1913. 

*-  For  discussion  of  formation  and  properties  of  these  two  ptomains,  see 
Vaughan  and  Xovy's  "Cellular  Toxins." 


590  GASTRO-INTESTINAL  "AUTOINTOXICATION" 

observed  in  the  feces  and  urine  of  persons  with  cystinuria.  The  stools  in  cholera 
also  seem  to  contain  these  ptomains  frequently.  Their  etiological; relation  to  the 
cystinuria  is  no  longer  accepted,  however,  and  their  toxicity  is  slight.  They  are 
probably  derived  from  the  diamino-acids  of  the  protein  molecule,  putrescine  being 
closely  related  to  ornithine,^^  and  is  probably  formed  from  it  as  follows: 

NH2  NH2  NH2  NH2 

i  '  I     ■ 

CH2-CH2-CH2-CH-COOH   =    CH2-CH2-CH2-CH2  +  CO2 

(ornithine)  (putrescine) 

while  cadaverine  is  probably  formed  from,  lysine, 
NH2  NH2  NH2  NH2 

CH2-(CH2)3-CH-COOH   =  CH2-(CH2)3-CH2   +  CO2 

(lysine)  (cadaverine) 

/NH2 
Ethvlidendiamine,    CH3 — CH<C  ^^„    which  is  somewhat  toxic,  has  also  been 

\NH2 

detected  in  the  contents  of  the  gastro-intestinal  tract. 

Apparently  these  substances  are  absent  from  normal  feces,  but  this  does  not 
exclude  the  possibility  of  their  normal  formation,  absorption  and  destruction. 
There  is  no  evidence  that  they  ever  cause  symptoms  or  pathological  alterations. 

(3)  Substances  Arising  from  the  Sulphur-Containing 
Radical  of  Proteins 

Most  if  not  all  of  the  sulphur  in  the  protein  molecule  seems  to  be  contained  in 
the  amino-acid,  cystine,  which  has  the  following  composition: 

S  -  CH2  -  CHNH2  -  COOH 

I 

S  -CH2  -CHNH2  -COOH. 

From  this  is  formed  the  hydrogen  sulphide  of  the  intestinal  gases,  of  which  about 
0.058-0.066  gram  is  present  in  each  one  hundred  grams  of  normal  colon  contents. 
Although  Senator  has  described  a  case  in  which  an  intoxication  with  US  of  in- 
testinal origin  occurred,  this  gas  seems  not  to  be  a  frequent  cause  of  intoxication, 
and  Senator's  case  stands  almost  alone.  Under  normal  conditions  H2S  does  not 
appear  in  the  urine,  any  that  may  be  absorbed  probably  being  oxidized  to  SO4.  If 
enough  H2S  should  enter  the  blood  so  that  it  was  not  completely  destroyed,  it 
might  well  cause  harm,  for  it  is  decidedly  toxic,  particularly  affecting  the  nervous 
system;  but  we  have  no  evidence  that  this  often  happens.  Van  der  Bergh'^  has 
observed  cases  of  intestinal  obstruction  in  which  the  presence  of  sulphernoglobin  in 
the  patient's  blood  was  demonstrated. 

Methyl  mercaptan,  CH3SH,  has  also  been  found  in  the  feces,  although  it  seems 
not  to  be  abundantly  or  constantly  present,  according  to  Herter,^^  who  found 
also  that  mixed  bacteria  from  normal  feces  rarely  produce  mercaptan  in  cultures. 
However,  bacteria  from  the  feces  of  persons  suffering  with  various  diseases  often 
produce  mercaptan.  Ethyl  mercaptan,  C2H5SH,  and  ethyl  stdphide,  C2H5-S-C2H6, 
have  also  been  described  as  fecal  constituents.  It  is  not  known  that  the  mercap- 
tans  are  a  cause  of  intoxication. 

Cystine  and  Cystinuria^s 

Cystine  has  been  observed  in  the  urine  in  a  number  of  cases,  and 

when  present  at  all  it  is  usually  found  in  considerable  quantities. 

*'  Ornithine  forms  part  of  the  arginine  molecule,  which  is  the  most  universally 
present  (in  proteins)  of  all  the  amino-acids,  ornithine  being  formed  when  urea 
is  split  from  arginine. 

"  Dent.  Arch.  klin.  Med.,  1905  (83),  8G. 

"  Jour.  Biol.  Chem.,  1906  (1),  421. 

88  Literature  concerning  cystine  given  by  Friedmann,  Ergebnisse  der  Physiol., 
1002  (I.  Abt.  1),  15;  and  l)y  Mann,  "Chemistry  of  the  Frotoins,"  ])p.  56-61.  Cys- 
tinuria reviewed  l)y  Bodtker,  Zcit.  ])hysiol.  Cliein.,  1905  (15),  39;?;  CJarnxl,  "Inborn 
Errors  of  Metabolism,"  and  Lancet,  July,  190S;  Kretsclimer,  Urol,  and  Cut.  Rev., 
1916  (20),  No.  1. 


(•)  STf.M':  AM)  (■)  STIMRIA  591 

Because  of  its  slight  sohihility  it  appears  as  a  deposit  of  hexagonal 
crystals,  and  frequentlj''  forms  cystine  concretions  (g.  v.)  in  the  urinary 
bladder."  According  to  Garrod  it  is  more  common  than  alkapto- 
nuria, and,  like  the  rest  of  the  "Inborn  Errors  of  Metabolism,"  occurs 
much  more  often  in  males  than  in  females.  Hofmann****  was  able  to 
collect  from  the  literature  to  1907  a  total  of  175  cases,  of  which 
85  were  males  and  45  females.  Baumann  and  others  observed 
that  in  cystinuria  the  urine  often  contains,  besides  the  cystine,  the 
diamines  cadavcrine  and  pntrescine,  which  are  formed  from  lysine 
and  ornithine  respectively  in  the  intestines  through  putrefaction, 
and  they  naturally  suspected  that  cystine  arose  in  the  same  way. 
Another  view  was  that  the  diamines  interfered  with  the  oxidation 
of  sulphur  in  the  body,  so  that  it  was  eliminated  in  the  unoxidized 
form  of  cystine.  But  it  has  been  demonstrated  that  neither  of  these 
hypotheses  is  correct,  for  (1)  cystine  could  not  be  found  in  the  feces; 
(2)  if  given  by  mouth,  it  is  completely  oxidized,  and  causes  only  the 
appearance  of  excessive  amounts  of  sulphates  in  the  urine;  (3)  cys- 
tinuria has  been  observed  to  occur  independent  of  the  presence  of 
the  diamines,  and  not  to  be  modified  or  caused  by  their  administration 
or  pathological  formation.  The  view  now  prevalent  is  that  the  cystine 
that  escapes  in  the  urine  in  cystinuria  is  not  derived  from  intestinal 
putrefaction,  but  is  formed  in  the  tissues  from  the  protein  molecule, 
and  fails  to  be  further  decomposed  because  of  some  anomaly  of  metab- 
olism. This  view  is  supported  by  the  fact  that  cystinuria  often  ap- 
pears to  be  an  hereditary  disease,  occurring  in  families  for  several 
generations,  it  is  independent  of  the  diet,  cystine  appearing  even  if 
proteins  are  withheld,  and  also  independent  of  intestinal  putrefac- 
tion.^^ It  having  been  found  that  leucine  and  tyrosine  may  also  occur 
in  the  urine  in  cystinuria,^*'  it  seems  probable  that  this  condition  de- 
pends upon  a  general  abnormality  of  protein  metabolism.  The  rela- 
tion of  the  diamines  to  the  condition  is,  however,  very  uncertain. 
Cystine  does  not  seem  to  exert  any  toxic  effect,  and  patients  with 
cystinuria  do  not  usually  appear  to  suffer  greatly  from  the  abnormal 
metabolism,  the  chief  trouble  observed  being  due  to  the  formation  of 
the  concretions  in  the  bladder.^^  Sometimes  in  children,  however, 
emaciation  and  earl}^  death  without  other  apparent  cause,  have  been 
observed,  and  may  be  due  to  the  metabolic  anomaly. 

The  metabolic  error  in  cystinuria  is  not  complete,  for  only  a  por- 
tion of  the  total  cystine  of  the  catabolized  proteins  is  excreted    as 

*'  Abderhalden  (Zeit.  physiol.  Chem.,  1903  (38),  557)  has  described  a  case  in 
a  child  in  which  the  organs  were  infiltrated  with  masses  of  the  cystine  crystals. 

88  Cent.  Grenz.  Med.  u.  Chir.,  1907  (10),  721. 

83  An  isolated  case  of  transient  cystinuria  in  a  patient  with  Raynaud's  disease 
is  described  bv  Githens  (Penn.  IMed.  Jour.,  1910  (1),  .507). 

90  See  Abderhalden,  Zeit.  physiol.  Chem..  1919  (104).  129. 

91  This  may  be  avoided  by  decreasing  the  cystine  by  means  of  a  low  protein 
diet,  and  increasing  its  solubilitj'  bv  keeping  the  reaction  of  the  urine  alkaline 
(Smillie,  Arch.  Int.  Med.,  1915  (16),  503). 


592  GASTRO-INTESTINAL  "AUTOINTOXICATION" 

such  (Garrod).  This  would  amount  to  some  five  grams  per  day, 
whereas  the  average  excretion  is  only  about  0.3-0.5  gram,  and  sul- 
phates and  other  neutral  sulphur  compounds  are  always  present  in 
the  urine.  In  no  condition  other  than  cj^stinuria  have  putrescine  and 
cadaverine  been  found  in  quantities  which  could  be  detected  by  ordi- 
nary methods  in  24-hour  specimens;  they  may  also  be  found  in  the 
feces  of  cystinurics,  where  cystine  is  never  found.  In  the  urine  their 
presence  is  inconstant,  and  the  amounts  are  at  best  very  small.  Leu- 
cine and  tyrosine  are  found  much  less  often  than  the  diamines;  lysine, 
has  been  found  in  one  case,^^  which  supports  the  view  that  cadaverine 
and  putrescine  come  from  the  diamino-acids  of  the  protein  molecule 
by  metabolism  rather  than  by  putrefaction. 

B.  Products  of  Fermentation  of  Carbohydrates 

These  include  practically  all  the  members  of  the  fatty  acid  series,  from  formic 
acid  to  valerianic  acid;  and  the  oxy-acids,  lactic,  succinic,  and  oxybutyric;  also, 
oxalic  acid,  acetone,  ethyl  alcohol,  and  the  following  gases:  CO2,  CH^,  H2.  For  the 
most  part,  the  various  organic  acids  are  absorbed  through  the  intestinal  walls,  and 
are  oxidized  completely  in  the  tissues  without  causing  any  harm  whatever.  The 
possibility  that  acid  intoxication  may  be  produced  in  this  way  has  been  suggested, 
but  it  is  generally  believed  that  this  does  not  occur,  except  possibly  in  infants. 
Lactic  and  butyric^^  acids  are  formed  particularly  in  gastric  fermentations  in  persons 
with  deficient  hydrochloric  acid,  motor  insufficiency,  or  organic  obstruction.  Most 
of  the  disturbances  observed  in  these  conditions  seem  to  be  due  to  distention  of  the 
stomach  with  gas,  chiefly  CO2,  which  is  formed  during  the  fermentation.  It  is 
possible,  however,  that  the  organic  acids  exercise  some  irritant  effects  on  the  mucous 
membrane;  and  they  may  also  cause  diarrhea,  lactic  and  acetic  acid  often  being 
present  in  diarrheal  discharges  due  to  excessive  feeding  with  carbohydrates  (Her- 
ter). 

These  acids  or  their  salts  do  not  appear  in  the  urine,  unless  possibly  as  minute 
traces,  except  the  oxalic  acid.  Minute  quantities  (0.02  gm.  per  day)  of  this  sub- 
stance are  present  in  normal  urine,  but  larger  quantities  {oxaluria)  seem  to  depend 
either  upon  the  taking  of  food  containing  much  oxalic  acid  (rhubarb,  spinach,  etc.) 
or  upon  excessive  gastric  fermentation  of  carbohydrates  (Baldwin),^''  and  perhaps 
upon  excessive  destruction  of  purines,  from  which  oxalic  acid  may  be  derived. 
Of  the  amino-acids  it  is  presumably  the  diatomic  acids,  glutamic  and  aspartic, 
which  yield  oxalic  acid  (Jastrowitz).^^  Others,  however,  do  not  admit  that  any 
appreciable  amount  of  oxalic  acid  is  derived  from  proteins. ^^  Probably  the  small 
quantities  of  oxalic  acid  thus  formed  do  not  cause  toxic  effects,  and  are  im- 
portant chiefly  as  causing  urinary  concretions  of  calcium  oxalate,  although  there  is 
evidence  that  long-continued  excretion  of  oxalic  acid  may  cause  renal  lesions. 
(See  also  consideration  of  oxalate  calculi,  Chap,  xvii.) 

C.  Products  of  the  Decomposition  of  Fats 

These  differ  but  little  in  nature  from  the  products  of  carbohydrate  fermenta- 
tion, the  large  fatty  acid  molecules  being  broken  down  to  smaller  ones.  In  infants 
these  fatty  acids  have  been  believed  to  be  a  cause  of  acid  intoxication  and  aceto- 
nuria,"  but  probably  they  are  seldom,  if  ever,  of  pathological  importance.     It  is 

"-  Ackcrmannand  Kutscher,  Zeit.  f.  Biol.,  1911  (57),  3.'55. 

"  Coleman  (Ann.  Inst.  Pasteur,  1915  (29),  139)  attempts  to  incriminate  butyric 
acid  in  the  i)roduction  of  arteriosclerosis,  while  Oswald  Loeb  believed  lactic  acid 
to  ha  important,  a  view  which  could  not  be  altogether  supported  by  Denny  and 
Frothingham,  .Jour.  Mod.  Res.,  1914  (31),  277. 

9"  Jour.  Exp.  Med.,  1900  (5),  27. 

"  Biochem.  Zeit.,  1910  (28),  34. 

»8  Wcgrzynowski,  Zeit.  physiol.  Chem.,  1913  (83),  112. 

07  Meyer  and  Langstein,  .Jahrb.  f.  Kinderheilk.,  1900  (03),  30. 


SIGNIFICANCE  OF  AUTOINTOXICATION  593 

possible,  however,  that  a  serious  reduction  in  the  bases  of  the  blood  may  result  from 
the  formation  of  excessive  amounts  of  fatty  acids  in  the  intestines,  the  bases  being 
coml)ined  to  unite  with  the  fatty  acids,  and  then  excreted  in  the  feces. 

It  is  quite  otherwise  with  the  jiroducts  of  decomposition  of  lecithin.*^  From 
its  molecule  is  split  off  the  ptomain,  choline, 

(CHa).,  s  N  -  CH,  -  CHjOII 

Ah 

which  is  easily  oxidized  into  a  highly  poisonous  compound,  isomeric  with  mus- 
carine, or  by  losing  a  molecule  of  water  it  forms  neuritiie, 

(CH3),  =  N— CH  =  CHi 

I 

OH 

which  is  also  very  poisonous  (discussed  under  "Ptomains,"  Chap.  iv.).  It  has 
been  demonstrated  by  Nesbitt^'  that  in  the  contents  of  obstructed  intestines  of 
dogs  that  have  been  fed  Iccithin-ricli  food  (eggs)  both  clioline  and  neurine  may  be 
found  free,  and  Kutscher  and  Lolimann'  have  found  neurine  in  human  urine.  It 
seems  possible  that  some  of  the  toxic  effects  observed  after  eating  excessively  of  such 
food  as  calves'  brains,  or  eggs,  may  depend  upon  intoxication  witli  the  products  of 
lecithin  decomposition.  Also,  the  normal  presence  of  trimethylanrx.ine  in  the  blood 
and  cerebrospinal  fluid  (Dorce  and  Golla)-  may  be  from  this  source.  Hunt,'  who 
has  done  extensive  work  with  choline,  states  that  at  present,  we  have  no  grounds  for 
believing  that  choline  has  any  significance  in  physiological  or  pathological  pro- 
cesses. There  is  no  evidence  that  the  highly  active  acetyl-cliolinc''  is  produced 
from  choline  in  the  bodj^,  but  in  view  of  the  enormous  toxicity  of  this  choline  de- 
rivative there  must  always  be  considered  the  possibility  that  such  toxic  choline 
compounds  may  at  times  develop  in  amounts  too  small  to  be  detected  but  large 
enough  to  cause  effects. 

Results  Of  Gastro-Intestional  Intoxication 

As  we  have  seen  from  the  above,  but  few  of  the  known  products  of 
gastro-intestinal  putrefaction  are  toxic  to  any  considerable  degree, 
and  these  are  probably  produced  in  too  small  quantities  to  cause  any 
appreciable  effect,  especially  in  view  of  the  detoxicating  and  eli mi- 
natory powers  of  the  liver,  kidney,  and  other  organs.  And  yet  we 
have  abundant  clinical  evidence  that  excessive  intestinal  putrefaction 
or  retention  of  the  intestinal  contents  causes  marked  disturbance  in 
health.  The  slight  malaise,  headache,  and  lassitude  observed  as  the 
result  of  simple  constipation  may  possibl}^  be  adequately  accounted  for 
by  intoxication  with  indole  and  similar  substances,  although  we  have 
no  conclusive  proof  that  such  is  the  case.  Two  explanations  may  be 
suggested :  One  is  that  the  intestinal  flora  becomes  altered  because  of 
the  changed  conditions,  and  bacteria  thrive  that  produce  specific 
soluble  toxic  substances,  analogous  to  those  formed  by  B.  hotuUtius, 
or  similar  to  the  hjrotoxicon  (Vaughan)  that  may  be  formed  in  milk 
and  milk  products.     Thus  Clairmont  and  Ranzi^  found  heat-resistant 

^*  Literature  given  by  Halliburton,  Ergebnisse  der  Phvsiol.,  190-4  (4\  24. 
"Jour.  Exp.  Med.,' 1899  (4),  1;  see  also  Hoesslin,  "Hofmeister's  Bcitr.,  1906 
(8),  27. 

'  Zeit.  phvsiol.  Chem.,  1906  (48),  1. 

-  Biochem.  Jour.,  1910  (5),  306. 

3  Jour.  Pharmacol.,  1915  (7),  301. 

^  See  Dale,  Jour.  Pharmacol.,  1914  (6),  147. 

6  Arch.  klin.  Chir.,  1904  (73),  696. 

38 


594  GASTRO-INTESTINAL  "AUTOINTOXICATION" 

toxic  substances  in  the  intestinal  contents  in  ileus  (experimental),  and 
similar  substances  could  also  be  obtained  by  growing  cultures  of  the 
intestinal  contents  on  bouillon.  Another  explanation  is  that  many 
unidentified  poisonous  substances  are  produced  in  the  alimentary 
canal  which  ordinarily  are  destroyed,  but  under  certain  conditions 
may  be  reabsorbed.  That  unrecognized  toxic  substances  are  formed 
in  the  intestines  is  almost  certain,  for  it  has  been  repeatedly  shown 
that  extracts  of  the  contents  of  the  alimentary  canal  are  very  poison- 
ous. Although  the  technic  of  many  of  these  experiments  has  been 
questionable,  the  results  have  been  obtained  so  often  as  to  render  it 
probable  that  the  main  contention  is  correct.^  Thus  Magnus-Alsle- 
ben^  has  found  in  the  upper  part  of  the  small  intestine  of  dogs  (ex- 
cept when  on  milk  diet)  a  very  poisonous  substance  which  killed  rab- 
bits by  respiratory  paralysis,  but  which  is  inert  when  injected  into  the 
portal  vein.  Extracts  of  the  wall  of  the  large  intestine  are  also  toxic, 
and  lose  their  toxicity  at  60°,  by  passing  through  porcelain  filters  and 
by  treatment  with  alcohol;  extracts  of  fetal  intestines  are  not  toxic 
(Distaso).^  There  is  reason  to  believe  that  histamine  is  responsible 
for  much  of  the  toxic  effects  obtained  with  intestinal  materials. 

In  any  case,  correctly  or  incorrectly,  a  great  number  of  disease  con- 
ditions have  been  attributed  to  poisons  of  gastro-intestinal  origin, 
including  not  only  such  minor  conditions  as  headache,  malaise,  lassi- 
tude, etc.,  but  also  sciatica,  tetany,  epilepsy,  eclampsia,  many  forms 
of  dermatitis,  various  forms  of  nervous  diseases,  myxedema  and 
cretinism,  chlorosis  and  pernicious  anemia,  cirrhosis,  nephritis,  and 
arteriosclerosis.^  While  in  many  cases  the  severity  of  these  various 
conditions  is  apparently  augmented  by  intestinal  disturbances,  the 
etiologic  relation  is  not  so  clear.  That  long-continued  intoxication  of 
intestinal  origin  may  cause  serious  injury  to  the  tissues  is,  however, 
extremely  probable.  There  is  much  reason  for  believing  that  many 
cases  of  non-alcoholic  cirrhosis  are  due  to  this  cause;  not  improbably 
chronic  nephritis,  myocarditis,  and  arteriosclerosis  may  occasionally 
be  the  result  of  long-continued  intoxication  from  the  same  source. 
Arteriosclerosis  especially  has  been  attributed  to  indole  and  related 
substances  by  Metchnikoff  and  his  associates,  who  have  produced  ar- 
teriosclerosis in  rabbits  by  injecting  indole,  but  not  with  skatole.  As 
is  well  known,  Metchnikoff  believed  that  most  of  the  manifestations  of 
senility  come  from  putrefaction  in  the  large  bowel, ^°  and  a  number 
of  observers  have  described  as  products  of  intestinal  putrefaction  cer- 
tain pressor  substances  of  high  potcnc,y  which,  presumably,  might 
cause  serious  arterial  and  cardiac  injury. ^^     An  elaborate  study  of 

*  For  example,  see  Roger  and  Gamier,  Compt.  Rend.  Soc.  Biol.,  1905  (59), 
388  and  674;  1906  (60),  666. 

'  Hofmeister's  Beitr.,  1905  (6),  503. 

8  Zcit.  Immunitat.,  1913  (16),  466. 

'  The  supposed  relation  of  gastro-intestinal  intoxication  to  these  various  dis- 
eases is  reviewed  by  Weintraud,  Ergeb.  allg.  Pathol.,  1897  (4),  17. 

'«  See  Ann.  Inst."  Pasteur,  1910  (24),  755. 

"  See  Granger,  Arch.  Int.  Med.,  1912  (10),  202. 


SIGMFICANCE  OF  AUTOI S TOMCAT  10 \  o'Jo 

a  certain  type  of  cases  of  defective  development  led  Herter'-  to  the 
conclusion  that  intestinal  intoxication  is  responsible,  and  hence  he 
designated  this  condition  "intestinal  infanlilisni." 

Tetany  associated  with  gastric  dilatation  ott'ers  perhaps  the  strongest 
case,  numerous  observers  having  reported  finding  a  marked  toxicity 
of  the  stomach  contents.'^  Pineles'''  considers  that  all  forms  of  tet- 
any, whether  of  gastric  origin  or  following  thyroidectomy,  are  due 
to  one  and  the  same  "tetany  poison,"  but  recent  studies  indicate  that 
alkalosis  is  an  important  factor  in  tetany.  There  is  also  considerable 
evidence  that  the  tetany,  when  present,  is  associated  with  a  deficiency 
in  calcium  in  the  blood  and  nervous  tissue,  and  that  this  is  further 
related  to  the  functional  activity  of  the  parathyroids  {q.  v.). 

Although  there  are  usually  evidences  of  intoxication  in  acute  dila- 
tation of  the  stomach,  yet  there  is  no  good  evidence  as  to  its  nature. 
It  is  suggested  by  Woodyatt  and  Graham^^  that  the  dilatation  is  pro- 
duced by  COo  secreted  into  the  stomach  from  its  walls. 

The  relation  of  intestinal  intoxication  to  the  various  anemias,  par- 
ticularly chlorosis  and  pernicious  anemia,  has  been  repeatedly  indi- 
cated and  discussed.  Clinical  evidence  strongly  indicates  that  such  a 
relation  exists,  and  there  is  no  doubt  that  hemolytic  substances  may 
be  formed  in  the  alimentary  tract, ^*^  but  that  chlorosis  and  pernicious 
anemia  do  depend  upon  intestinal  putrefaction  or  infection  is  far 
from  established  (see  "Anemia,"  Chap.  xiii). 

As  yet,  however,  we  cannot  say  positively  that  any  human  disease  is 
caused  by  the  products  of  intestinal  putrefaction,  and  with  growing 
knowledge  the  importance  ascribed  to  this  source  of  disease  is  becom- 
ing steadily  less.^'  The  fact  must  not  be  overlooked  that  many 
persons  habitually  eat  putrefied  proteins,  from  the  "high"  game  of 
the  epicure  to  the  carrion  masses  that  dehght  many  primitive  people, 
without  the  shghtest  evidence  of  intoxication  therefrom. 

It  seems  highly  probable  that  gastro-intestinal  "autointoxication" 
would  be  a  much  more  serious  matter  were  it  not  for  the  mechanisms 
of  defence  possessed  by  the  body,  especially  in  the  liver. '^  For  exam- 
ple, Richards  and  Rowland  have  indicated  the  increased  toxicity  of 
indole  when  the  oxidizing  power  of  the  liver  is  reduced,  and  Herter 
and  Wakeman  have  shown  the  power  of  the  liver  to  combine  indole 
and  thus  remove  it  from  circulation.  This  topic  has  been  discussed 
more  fully  elsewhere  (Chap,  x) . 

12  See  McCrudden,  Jour.  Exper.  Med.,  1912  (15),  107. 

1'  Bibliography  by  Halliburton  and  McKendrick,  Brit.  Med.  Jour.,  1901  (i), 
1607. 

1*  Deut.  Arch.  klin.  Med.,  1906  (85),  491. 

'5  Trans.  Chicago  Path.  Soc,  1912  (8),  354. 

'«  See  Kiilbs,  Arch,  exper.  Path.,  1906  (55),  73;  also  Herter,  Jour.  Biol.  Chem., 
1906  (2),  1. 

1^  See  Alvarez,  Jour.  Amer.  Med.  Assoc,  1919  (72),  8;  E.  O.  Jordan,  "Food 
Poisoning,"  Univ.  Chicago  Press.  1917. 

18  For  discussion  and  literature  see  Lust,  Hofmeister's  Beitr.,  1905  (6),  132. 


596  GASTRO-INTESTINAL  "AUTOINTOXICATION" 

Acute  Intestinal  Obstruction 

The  violent  effects  that  follow  complete  occlusion  of  the  intes- 
tine, especially  in  the  upper  portion,  must  be  due  to  some  highly  toxic 
substance  or  substances.  The  clinical  features  of  obstructive  ileus, 
namely,  vomiting,  collapse,  complete  muscular  relaxation,  and  sub- 
normal temperature,  are  associated  with  the  excretion  of  large  quan- 
tities of  indican  and  other  substances  combined  with  sulphuric  acid, 
proving  that  intestinal  putrefaction  is  active.  Undoubtedly  in  ileus 
we  have  a  profound  and  rapidly  fatal  intoxication  with  substances 
formed  in  the  obstructed  intestines. 

Whipple^^  has  demonstrated  that  closed  duodenal  loops  in  dogs 
come  to  contain  a  highly  toxic  substance  of  unknown  nature,  appar- 
ently formed  in  the  epithelium  of  the  gut  rather  than  in  its  contents, 
which  causes  severe  splanchnic  congestion,  vomiting  and  diarrhoea 
when  injected  into  normal  dogs.  The  toxic  agent  is  not  destroyed  by 
autolysis,  filtration  or  heating  at  60°,  yet  dogs  can  be  made  somewhat 
refractory  or  immune.  From  the  contents  of  such  loops,  and  from  the 
bowel  above  obstructions,  he  has  isolated  a  very  toxic  proteose,-"  which 
he  believes  may  be  responsible  for  the  intoxication.  Whether  this 
proteose,  or  whatever  the  active  poison  may  be,  comes  from  bacterial 
infection,  autolysis,  duodenal  secretion,  or  what,  is  not  yet  agreed  by 
the  numerous  investigators  in  this  field. -^  There  is  much  evidence  in 
favor  of  the  essential  importance  of  bacteria,  and  it  is  held  by  some 
that  toxic  amines  may  be  produced  by  bacterial  action  in  the  closed 
loops, ^"  and  that  injury  to  the  mucosa  facilitates  absorption  of  the 
poisons.  Apparently  the  liver  does  not  have  much  detoxicating  effect, 
for  dogs  with  Eck  fistula  behave  much  the  same  when  the  intestine  is 
obstructed  as  dogs  with  normal  circulation.  A  similar  material  can- 
not be  obtained  by  hydrolysis  or  autolysis  of  normal  duodenal  mucosa, 
the  obstruction  being  an  essential  feature.  The  normal  intestinal 
secretions  do  not  have  any  considerable  toxicit3^-^  Obstruction  of 
lower  portions  of  the  intestine  has  much  less  effect'^  and  it  has  been 
suggested  that  the  poison  formed  in  the  duodenum  is  neutralized  or 
destroyed  farther  down  in  the  intestine.-^  A  striking  feature  of 
intestinal  obstruction  is  the  high  non-protein  nitrogen  content  of  the 
blood,  figures  similar  to  those  of  fatal  uremic  coma  being  common,-^ 
which  may  be  the  result  of  absorption  of  cleavage  products  from  the 
intestine,  or  toxicogenic  destruction  of  tissue  proteins,  or  both.  There 
is  also  pro1)ably  an  element  of  renal  injury  and  reduced  excretion.-^ 

'»  Whipple,  Stone  and  Bernheim,  Jour.  Exp.  Mod.,  1913  (17),  280. 
-"Jour.  Aiuer.  Med.  Assoc,  1915  (G5),  470;  1910  (07),  15;  Jour.  Exp.  Med., 
1910  (23),  123;  1917  (25),  231  and  401. 

2^  Review  hv  South  and  Hardt,  Arcli.  Int.  Med.,  191S  (21),  292. 

■^~  Drajistedt  et  nl.,  Jour.  Exp.  Med.,  1919  (30),  109. 

"  Davis  and  Stone,  Jour.  Exp.  Med.,  1917  (20),  OSO. 

■■"See  BuiitiiifT,  Jour.  ]<;xi).  Med.,   1913  (17),  192. 

"  Maurv,  Ainer.  Jour.  Med.  Sei.,   1909  (137),  725. 

"  Cooke,  Hodenhaumh  and  Whipple,  Jour.  Exp.  Med.,  1910  (23),  123. 

"McQuarric  and  W  liipple.  Jour.  Exp.  Med.,  1919  (29),  397. 


("HAPTi:il  XXII 

CHEMICAL  PATHOLOGY   OF  THE  DUCTLESS   GLANDS' 

DISEASES  OF   THE  THYROID- 

As  we  have  much  evidence  that  the  thyroid  has  a  marked  infhieiice 
upon  metabohsm,  and  also  that  it  may  be  of  imp^ortance  in  preventing 
and  in  producing  autointoxication,  the  chemistry  of  diseases  of  the 
thyroid  may  be  appropriately  considered  in  connection  with  the  auto- 
intoxications. 

The  Functions  of  the  Thyroid 

Metabolic  Function. — That  the  thyroid  has  an  impoitant  rela- 
tion to  metabolism,  especially  of  proteins,  is  shown  by  the  following 
facts: 

(1)  Administration  of  the  gland  substance,  or  active  preparations  made  from 
it,  to_  healthy  men  or  animals,  causes  a  greatly  increased  elimination  of  nitrogen  in 
the  form  of  area."  This  nitrogen  comes  not  only  from  the  food,  but  also  from  in- 
creased tissue-destruction,  as  is  shown  by  the  loss  of  weight  and  strengtli,  and  by 
the  increased  excretion  of  sulphur  and  phosphorus.  An  increased  destruction  of 
the  body  fat  also  occurs,  so  that  thyroid  therapy  has  been  found  efficient  in  the 
treatment  of  obesity,  but  often  dangerous  because  of  the  relatively  great  amount 
of  tissue-destruction.  Basal  metabolism  is  most  markedly  raised  in  hyperthyroi- 
dism, and  is  lower  in  cretinism  and  myxedema  than  in  any  other  disease* 

(2)  Loss  of  thyroid  tissue,  either  through  operation  or  disease,  greatly  reduces 
both  nitrogenous  metabolism  and  oxidative  processes.  Administration  of  thyroid 
preparations  under  these  conditions  will  bring  the  nitrogen  elimination  and  the 
gas  exchange  back  to  normal. 

(3)  Dehcient  thj-roid  secretion  in  3'oung  animals  prevents  their  developing  nor- 
mally, the  amount  of  deficiency  varying  from  nearly  total  lack  of  development  in 
extreme  cretinism  to  slight  grades  of  defective  development  (infantilism)  or  de- 
layed maturity.  In  adult  animals,  besides  decreased  metabolism  there  occur  also 
various  trophic  changes  in  the  skin  and  its  appendages,  an  increased  amount  of 
mucin-Iike  material  in  the  tissues,  and  greatly  decreased  nerv^ous  and  mental  ac- 
tivity. All  these  conditions  are  relieved  to  greater  or  less  degree  by  administration 
of  thyroid  tissue  or  its  preparations.^  Evidently,  therefore,  the  thyroid  exerts  an 
influence  upon  growth  and  tissue  changes;  whether  this  depends  upon  its  influence 
upon  metabolism,  or  is  an  independent  and  specific  function,  cannot  be  deter- 
mined.^ 

^  Thorough  reviews  of  the  entire  subject  of  the  ductless  glands  are  given  by 
Biedl,  "Innere  Sekretion,"  Urban  and  Schwarzenberg,  Berlin,  11)13;  and  Vincent, 
Ergebnisse  Physiol.,  1910  (9),  451;  1911  (10),  218. 

-  Concerning  the  thyroid  see  besides  Beidl  and  Vincent,  the  review  bv  Bircher, 
Ergebnisse  Pathol.,  1911,  XV  d),  82;  Crotti,  "'Thyroid  and  Thymus,"  Phila.,  1918. 

*  See  Rohde  and  Stockholm,  Jour.  Biol.  Chem.,  1919  (37),  305. 

*  Du  Bois,  Arch.  Int.  Med.,  1916  (17),  915. 

*  Concerning  the  influence  of  thyroid  on  skeletal  growth  see  Holmgren,  X'ordiskt 
Med.  Arkiv,  1910  (43),  No.  2.  Literature  given  by  Basinger,  Arch.  Int.  Med., 
1916  (17),  260. 

^  See  the  interesting  experiments  of  Gudernatsch  (Arch.  Entwickl.,  1912  (35), 
457;  Amer.  Jour.  Anat.,  1914  (15),  431;  Anat.  Record,  1917  (11),  357),  who  found 
that  feeding  thyroid  to  tadpoles  hastens  their  differentiation  but  checks  growth; 
also  removal  of  the  thyroid  from  tadpoles  increases  growth  but  delays  or  entirely 
prevents  metamorphosis  (Hoskins,  Jour.  Exp.  Zool.,  1919  (29),  1). 

597 


598         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

How  the  thyroid  or  its  secretion  modifies  metabolism  is  not  yet  understood. 
One  is  reminded  of  the  effects  of  kinases  upon  enzymes  and  their  antecedents, 
and  it  may  he  imagined  that  the  th\roid  secretion  activates  both  proteolytic  and 
oxidative  "enzymes  within  the  cells.  Shryver,'  indeed,  did  find  that  administra- 
tion of  thyroid  to  dogs  for  some  time  lief  ore  killing  them  causes  their  li\-er  tissue 
to  undergo  autolysis  more  rapidly  than  normal,  although  Wells*  had  been  unable 
to  observe  any  increased  amount  of  autolysis  when  thyroid  extracts  acted  upon 
liver  tissue  in  vitro.  Experimental  observations  show  that  carbohydrate  meta- 
bolism is  much  influenced  by  the  thyroid,  so  that  thyroidectomized  animals  may 
fail  to  show  glycosuria  from  various  procedures  that  usually  produce  it  (King),^ 
and  they  are  incapable  of  utilising  sugar  injected  parenterally  as  well  as  normal 
animals;^"  they  also  exhibit  an  excessive  creatine  output.  Protracted  feeding  of 
thyroid  to  growing  animals  reduces  the  weight  attained  and  causes  marked  en- 
largement of  suprarenals,  heart,  liver,  spleen,  testes,  ovaries  and  pancreas;  the 
pituitary  and  uterus  are  reduced  in  size." 

Detoxicatory  Function. — The  evidence  that  the  thyroid  has  for 
its  function  the  destruction  or  neutralization  of  poisonous  substances 
formed  in  metaboHsm  or  through  intestinal  putrefaction  is  as  follows: 

(1)  After  total  removal  of  the  thyroid  from  many  species  of  animals  acute 
symptoms  develop  that  suggest  strongly  an  intoxication. 

(2)  After  removal  of  the  thyroid,  marked  changes  occur  in  the  blood,  there 
being  a  severe  anemia  (as  low  as  2,000,000  red  corpuscles),  with  some  leucocytosis, 
and  there  occur  structural  changes  in  the  blood-vessel  walls  (Ivishi).'^  Cyto- 
plasmic degeneration  of  the  liver,  kidneys,  and  myocardium  may  also  result  (Ben- 
sen).  ^^  These  effects  suggest  strongly  ^he  presence  of  poisonous  substances  in  the 
blood  of  persons  or  animals  lacking  sufficient  thyroid  tissue. 

(3)  All  the  effects  of  thyroidectomy  are  more  marked  in  carnivorous  animals 
than  in  herbivora;  indeed,  the  latter  may  be  able  to  live  in  fair  condition  for  several 
years  without  a  thyroid."  Administration  of  meat  to  thyroidectomized  herbi- 
vora or  omnivora  causes  a  great  increase  in  the  symptoms,  while  thyroidectomized 
carnivora  do  much  better  if  kept  without  meat.  Thus,  Blum'^  found  that  thy- 
roidectomized dogs,  which  \\ere  doing  well  on  a  milk  diet,  developed  symptoms 
of  athyreosis  immediately  they  were  given  meat.  This  fact  has  been  interpreted 
as  indicating  that  toxic  materials  are  formed  from  meat  in  the  intestinal  tract, 
which  under  normal  conditions  are  neutralized  by  the  thyroid.  On  the  other 
hand,  one  may  well  imagine  that  the  so-called  autointoxication  in  athyreosis  is 
not  from  intestinal  putrefaction,  but  is  due  to  the  products  of  incomplete  meta- 
bolism of  proteins  within  the  tissues,  which  are  destroyed  when  protein  metabolism 
is  normal,  but  not  when  the  metabolism-favoring  influence  of  the  thyroid  is  want- 
ing. It  should  also  be  added  that  the  presence  of  specific  poisonous  substances  in 
the  blood  or  urine  of  thyroidectomized  animals  has  not  been  conclusivelj'  estab- 
lished.'" 

7  Jour,  of  Physiol,  1905  (32),  159. 

*  Amer.  Jour.  Physiol.,  1904  (11),  351;  corroborated  bv  Morse,  Jour.  Biol. 
Chem.,  1915  (22),  125. 

»  Jour.  Exper.  Med.,  1909  (11),  6G5. 

10  Underbill  and  Saiki,  Jour.  Biol.  Chem.,  1908  (5),  225. 

"Herring,  Quart.  Jour.  Exp.  Physiol.,  1917  (11),  231;  Kojima,  ibid.,   p.  255. 

12  Virchow's  Arch.,  1904  (176),  260. 

"  Virchow's  Arch.,  1902  (170),  229. 

^*  Part  of  these  results  may  be  due  to  the  fact  that  in  some  herbivora  tlie 
parathyroids  are  so  far  separated  from  the  thyroid  that  they  are  not  ordinarily 
removed  in  thyroidectomy,  whereas  in  many  carnivora  complete  removal  of 
parathyroids  with  the  thvroids  is  more  likely  to  be  accomplished. 

1"  Virchow's  Arch.,  1900  (162;,  375. 

I'Remedi  (Lo  Sperimentale,  1902;  abst.  in  Cent.  f.  Path.,  1903  (14),  695) 
claims  that  tetanus  toxin  and  other  bacterial  poLsons,  when  injected  into  the 
thyroid  gland,  are  harmless,  whicli  he  attributes  to  n  ncutraliziition  by  tlie 
colloid.  This  observation  is  discredited  by  the  work  of  Basinger,  Jour.  Infect. 
Dis.,  1917  (20),  131. 


CHEMISTRY  OF  TlIK  THYROID  599 

(4)  Reid  Hunt''  found  tliiit  mice  fed  thyroid  preparations  have  a  greatly  in- 
creased resistance  to  poisoning  l)yaceto-nitrile;  however,  this  is  not  necessarily  nor 
even  probably  a  direct  detoxication,  but  more  likely  it  results  from  alterations  in 
metabolism.'"  Rats  and  Ruinca  pigs  behave  just  the  opposite,  showing  a  decreased 
resistance  to  acetonitrile  after  being  fed  thyroid,  and  according  to  some  authors 
morphine  is  more  toxic  for  such  animals." 

Whether  the  thyroid  exercises  its  detoxicating  effect,  assuming  that 
it  has  one,  by  a  direct  neutrahzing  action  of  its  secretion  upon  the 
to.xic  substances  in  the  blood  or  in  diver.se  tissues,  or  indirectly  by 
stimulation  of  the  function  of  other  tissues  which  perform  the  detoxica- 
tion, or  in  part  locally  within  the  gland  itself,  is  an  unsettled  problem. 
In  relation  to  the  last-named  hypothesis  is  the  extreme  vascularity 
of  the  thyroid,  which,  according  to  Burton-Opitz-"  has  passed  through 
it  much  more  blood  in  proportion  to  its  weight  than  any  other  gland. 
Against  the  idea  of  a  local  detoxication  is  the  fact  that  after  extirpation 
of  the  thyroid  all  abnormal  conditions  may  be  prevented  by  proper 
administration  of  thyroid  substance. 

Biedl  summarizes  his  views  as  to  the  function  of  the  thjToid,  in 
the  following  statement:  "The  thyroid  is  a  secretory  organ  which 
discharges  its  secretion  eventually  into  the  blood,  in  the  form  of  an 
iodin-containing  protein.  This  secretion  acts  as  a  hormone,  in  that 
it  modifies  the  activities  of  remote  tissues.  As  far  as  we  now  know 
the  thyroid  secretion  plays  the  role  of  a  'disassimilatory'  hormone,  in 
that  it  causes  an  increased  disassimilation  and  increase  of  normal  activ- 
ity in  many  tissues.  This  effect  is  exemplified  by  the  augmented 
metabolism,  the  activity  of  the  heart  and  many  parts  of  the  sympa- 
thetic nervous  system,  and  of  a  series  of  internal  secretory  organs 
(adrenals,  hypophysis).  In  other  tissues  are  found  evidence  of  the 
action  of  an  inhibiting  and  assimilatory  hormone,  as  shown  in  the  in- 
crease in  growth  of  bone,  development  of  the  sex  glands,  and  decreased 
internal  secretion  of  the  pancreas." 

Chemistry  of  the  Thyroid^' 

Whether  the  function  of  the  thyroid  is  the  neutralization  of  toxic 
substances,  or  a  complementary  action  upon  intracellular  metabolism, 
there  can  be  little  question  that  it  owes  its  action  to  constituents  of 
its  specific  secretion,  the  colloid.--  Furthermore,  the  chief,  if  not  the 
sole,  active  ingredient  of  the  colloid  is  the  iodin-containing  substance 

I''  Jour.  .\mer.  Med.  Assoc,  1907  (49),  240;  Hygienic  Lab.  BuU.,  1909,  No.  47; 
Jour.  Pharmacol.,  1910  (2),  15. 

1*  Koopman  (Endocrinology,  1919  (3),  318)  states  that  administration  of 
thyroid  increases  antibody  formation  in  immunized  animals. 

19  See  Olds,  Amer.  Jour.  Phvsiol.,  1910  (26),  354. 

-0  Quart.  Jour.  Physiol.,  1910  (3),  297. 

21  Reviews  are  given  by  Rahel  Hirsch,  Handb.  d.  Biochem.,  1909,  III  (i), 
271;  and  A.  Kocher,  Virchow's  Arch.,  1912  (208),  86. 

--  Beyond  the  characteristic  colloid  secretion  product,  the  thyroid  presents  no 
chemical  features  of  interest;  it  differs  from  the  other  endocrine  glands  in  being 
poor  in  lipoids  (Fenger,  Jour.  Biol.JChem.,  1916  (27),',303). , 


600         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

first  discovered  by  Baumann  in  1896,  and  called  by  him  thyroiodin 
(or  iodothyrein)  J^ 

The  chemical  nature  of  thyroid  colloid  has  been  studied  particularly 
by  A.  Oswald.--*  He  found  that  all  the  iodin  of  the  thyroid  is  dissolved 
out  in  physiological  salt  solution,  and  that  none  of  it  is  present  in  an 
inorganic  form.  In  the  salt  solution  extract  are  two  protein  bodies; 
one,  precipitated  by  half  saturation  with  ammonium  sulphate,  con- 
tains all  the  iodin,  and  seems  to  be  a  globulin;  it  resembles  myosin 
in  being  precipitated  by  weak  acids,  and  it  contains  an  easily  separated 
carbohydrate  group.  The  other,  precipitated  by  saturation  with  am- 
monium sulphate  (exact  limits  of  precipitation  are  between  6.4  and 
8.2  tenths  saturation),  is  a  nucleoprotein,  containing  0.16  per  cent, 
phosphorus,  but  no  iodin;  it  is  without  marked  physiological  activity 
as  also  is  the  protein-free  watery  extract  of  the  thyroid.-^  The  col- 
loid seems  to  contain  practically  all  the  iodin  present  in  the  gland 
(Tatum).26 

The  iodin-containing  protein,  called  thyreoglohulin,  constitutes  one- 
fourth  to  one-half  the  dry  weight  of  the  gland,  and  seems  to  contain 
the  sole  active  constituent  of  the  colloid;  at  least,  its  administration  to 
animals  has  the  same  physiological  effects  as  does  the  entire*  colloid 
(great  increase  in  the  urea  elimination  and  decrease  in  blood  pressure 
in  animals,  curative  effect  on  myxedematous  patients,  increased  tonus 
of  both  sympathetic  and  peripheral  nervous  systems.)-'^  Analysis 
of  the  thyreoglobulin  from  various  animals  has  shown  it  to  be  of  quite 
constant  quantitative  composition  except  for  the  iodin,  which  may 
vary  greatly  in  amount.  Normal  human  thyreoglobulin  (from  persons 
living  in  non-goitrous  districts)  has  the  following  percentage  compo- 
sition : 

C  =  51.85,  H  =  6.88,  N  =  15.49,  I  =  0.34,  S  =  1.86. 
Thyreoglobulin  from  goitrous  districts  contains .  much  less  iodin 
(0.18-0.19  per  cent.),  and  from  calves  born  with  goiters  a  thyreo- 
globulin was  obtained  that  agreed  in  all  respects  with  normal  thrj-reo- 
globulin,  except  that  it  contained  no  iodin  at  all.  On  the  other  hand, 
administration  of  iodides  to  patients  causes  the  thyreoglobulin  to 
become  rich  in  organically  bound  iodin. ^**     From  these  facts  Oswald 

^'  Iodin  is  present  in  the  thyroid  of  all  species,  most  in  marine  forms  (Cam- 
eron, Jour.  Biol.  Chem.,  1914  (16),  465;  Biochem.  .lour.,  1014  (7),  466). 

'^"^  His  work  is  reviewed  in  his  dissertation,  "Die  chem.  Beschaffenheit  und  die 
Function  der  Schilddruse,"  Strassburg,  1900;  also  see  Vircliow's  Arch.,  1902 
(169),  444. 

^^  A.  Oswald,  Arch.  ges.  Physiol.,  1916  (166),  169.  Kocher  and  his  collabo- 
rators, however,  ascribe  to  the  thyreonucleoprotein  some  slight  metabolic  olTects 
antagonistic  to  the  thvreoglobulin  (See  Mitt.  Crenz.  Med.  Chir.,  1916,  vol.  29). 

2"  Proc.  ;Soc.  Kxp.  Biol.  Med.,  1919  (17),  28. 

"  A.  Oswald,  Arch.  ges.  Plivsiol.,  1916  (164),  506. 
_  2«  Nagel  and  Roos  (Arch.  f.  iVnat.  u.  Physiol.,  1902,  p.  297)  found  tliat  ad- 
ministration of  ))roini(les  had  no  effect  upon  the  amount  of  iodin  in  the  thyroid, 
and  no  storage  of  brornin  takes  place.     Administration  of  pilocarpine  docs  not 
increase  the  amount  of  iodin  in  the  thyroid. 


CHE^fISTRy  or  tiik  'nivh'oiD  ooi 

believes  that  the  thjTeofflobulin,  u.s  first  secreted  by  the  ^daiuhihir  epi- 
thelium, is  free  from  iotlin,  and  that  it  combines  later  with  iodin  from 
the  circulating  blood.  Thyro()gl()l)ulin  is  not,  however,  simply  an 
iodized  protein,  for  the  iodized  proteins  that  can  be  artificially  i)repared 
do  not  possess  the  physiological  activity  of  the  thyreoglobulin,  nor 
do  other  naturally  occuring  iodin-containing  proteins  (gorgonin, 
spongin).  F.  C.  Koch-'-*  finds  that  the  full  activity  of  the  gland  is 
contained  in  the  thyreoglobulin,  and  also  in  the  metaprotein  fraction 
of  this  gloinilin,  while  simpler  cleavage  products  show  less  and  less 
activity  per  unit  of  iodin  content.  He  could  find  no  thyroid  activity 
in  iodin  compounds  of  histidine,and  di-iodotyrosinewasfounrl  inactive 
by  Strouse  and  Voegtlin.^" 

The  remarkable  influence  of  the  thyroid  on  the  development  of 
tadpoles  (Gudernatsch")  is  exhibited  by  the  thyreoglobulin  but 
also  by  iodized  blood  proteins. ^^  Swingle'^^  states  that  inorganic  iodin 
itself,  even  in  thyroidless  tadpoles,  will  bring  about  metamorphosis 
that  would  not  take  place  in  the  absence  of  iodine.  Bromine  will  not 
substitute  for  iodin  in  causing  metamorphosis.  The  division  rate  of 
even  unicellular  organisms  (/''a /•a?«.eaww)  is  increased  by  thyroid  ex- 
tract. ^^  Lenhart^''  considers  the  effect  of  thyroid  on  tadpoles  to  be 
merely  an  expression  of  the  general  stimulation  of  metabolism,  rather 
than  a  specific  effect  on  differentiation.^^ 

By  decomposing  thyreoglobulin  by  boiling  with  10  per  cent, 
sulphuric  acid,  a  body  is  obtained  containing  as  high  as  14.5  per 
cent,  of  iodin;  this  is  the  thyroiodin  of  Baumann,  which  gives  no 
biuret  reaction,  yet  is  physiologically  active.  The  stability  of  this 
active  constituent  of  the  thyreoglobulin  explains  the  successful  ad- 
ministration of  thyroid  preparations  by  mouth.  It  appears  to  be 
absorbed  unchanged  and,  unless  enormous  doses  are  given,  none  ap- 
pears in  the  urine  (Oswald). ^^  Long-continued  digestion  with  trj'psin, 
or  autoh'sis  of  th3-roid  glands,  causes  a  complete  splitting  out  of  the 
iodin.  One  part  of  the  iodin  seems  to  be  more  firmh'  bound  than  the 
rest.  A  small  amount  of  the  iodin  may  exist  as  inorganic  and  lipoid 
compounds.  ^^ 

Thyroxin. — ^KendalP^  has  isolated  from  the  thyroid,  after  alkaline 
hydrolj'sis,  a  crystalline  compound  containing  65  per  cent,  of  iodin 
which  seems  to  be  an  indole  derivative,  designated  as  "thyroxin" 

23  Jour.  Biol.  Chem.,  1913  (14),  101. 

30  Jour.  Pharm.  and  Exp.  Ther.,  1910  (1).  123. 

31  Rogoff  and  Marine,  Jour.  Pharm.,  1917  (10),  321. 

'2  Jour.  Gen.  Phvsiol.,  1919  (1),  593;  Jour.  E.\p.  Zool.,  1919  (27),  397. 

"  Shumway,  Jour.  Exp.  Zool.,  1917  (22),  529. 

3^  Jour.  Exp.  Med.,  1915  (22),  739. 

35  See  also  Kahn,  Arch.  ges.  phvsiol.,  1916  (163),  384. 

3«  Arch.  exp.  Path.  u.  Pharm.,  1910  (63),  263. 

3"  Blum  and  Grutzner,  Zeit.  phvsiol.  Chem.,  1914  (91),  400. 

38  Jour.  Amer.  Med.  Assoc,  1918  (71),  871;  Trans,  .\ssoc.  Amer.  Phvs.,  1918 
(33),  324;  Endocrinology,  1919  (3),  156;  Jour.  Biol.  Chem.,  1919  (39),  125;  1919 
(40),  265. 


602         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

(thyro-oxy-indole)  with  the  following  structural  formula  which  has 
been  confirmed  by  its  synthetic  production: 

HI 

HI  1^  C— CH2— CH2— COOH 


HI 


\^-g-co 


The  activity  of  this  substance  is  referable  to  the  CO-NH  groups, 
since  acetylating  the  imino  group  removes  the  physiological  effect. 
In  the  body  the  indole  ring  seems  to  exist  in  the  open  form  shown 
below,  corresponding  to  the  open  and  closed  structure  of  creatine  and 
creatinine. 

HI 
HI  /\  C— CH2CH2COOH 

HI  I       I  COOH 

\^\NH2 

This  substance  is  highly  active,  causing  rapid  pulse,  nervous 
irritability,   and  increased  metabolism. 

Through  its  action  on  the  metabolism  of  all  the  cells  of  the  body 
thyroxin  relieves  all  the  manifestations  of  myxedema  and  cretinism. 
It  is  estimated  that  one-third  milligram  increases  the  basal  metabolism 
of  an  adult  by  1  per  cent.,  on  which  basis  it  may  be  calculated  that 
from  23  to  50  mg.  are  functioning  in  the  entire  body.  It  acts  like  a 
true  catalyst  in  carrying  on  action  for  two  or  three  weeks  after  being 
administered.  It  contains  only  one-fourth  of  the  iodin  of  the  thyroid 
but  the  nature  of  the  other  iodin  compounds  is  unknown,  bej^ond 
the  observation  that  they  have  no  appreciable  effects  on  normal 
persons  but  greatly  improve  the  condition  of  cretins,  presumably  they 
represent  intermediate  stages  in  the  formation  of  thyroxin.  AVhen 
fed  to  tadpoles,  Kendall's  active  principle  produces  the  characteristic 
thyroid  effect. ^^ 

Persons  with  complete  atrophy  of  the  thyroid  have  a  basal  meta- 
bolism 60  per  cent,  of  normal,  which  can  be  brought  to  normal  by  thy- 
roxin, but  what  carries  the  60  per  cent,  metabolism  when  the  thyroid 
is  functionless  is  unknown.  Presumably  thyroxin  merely  increases 
the  rate  of  metabolism  that  goes  on  at  a  lower  level  without  it,  in 
which  case  the  function  of  the  thyroid  seems  to  be  merely  to  permit  a 
greater  range  of  flexibihty  in  energy  output.  A  single  large  injection 
has  little  effect  while  repeated  small  doses  cause  death,  indicating  that 
thyroxin  of  itself  is  not  toxic,  but  only  through  its  effect  on  metabolism. 
The  limit  of  tolerance  is  2  mg.  daily. 

Iodin  Content  of  Thyroid. — The  physiological  activity  of  thyroid 
preparations,  according  to  nearly  all  investigators,  is  in  direct  propor- 

"  Rogoff  andMarine,  Jour.  Pharmacol.,  1916  (9),  57. 


CHEMisriivoF  THE  riiYuon)  dos 

tion  to  the  iotliii  content,'"  wliich  is  tlie  best  of  evidence  that  tlie  for- 
mation of  this  compound  is  one  of  the  chief  functions  of  the  gland, 
and  that  the  iodin  in  the  thjToid  is  not  merely  stored  there  as  an  unde- 
sirable foreign  substance  like  copper  in  the  liver.  The  selective  de- 
position of  iodin  in  the  tiiyroid  is  remarkable,  and  when  iodin  is  fed 
to  animals  it  is  stored  very  rapidly  but  it  seems  to  require  several 
hours  before  the  active  growth-modifying  hormone  is  formed.'"  Ma- 
rine and  Lenharf-  find  that  the  normal  human  j^land  contains  an  aver- 
age of  0.4  mg.  of  iodin  per  gram  of  fresh  weight  (2.17  mg.  per  gram 
of  dry  weight),  being  less  than  that  of  domestic  animals  in  the  same 
part  of  the  country.  These  figures  agree  closely  with  those  obtained  in 
thyroids  from  various  parts  of  America  by  Wells'*'  (2.10  mg.  per  gram 
dry  weight).  They  found,  as  Oswald  and  Kocher  also  have,  that  the 
amount  of  iodin  varies  directly  with  the  amount  of  colloid,  being  de- 
creased when  cellular  hyperplasia  is  present,  in  direct  proportion 
to  the  amount  of  hyperplasia,  and  administration  of  iodin  causes  a 
reduction  in  the  hyperplasia  and  a  return  to  the  colloid  type  of  gland, 
while  the  iodin  is  deposited  in  the  gland.  Kocher,  however,  disputes 
the  regularity  of  the  variation  of  iodin  and  colloid  content,  stating 
that  it  is  especially  the  concentrated  follicle  contents  which  hold  the 
iodin.  Seidell  and  Fenger'*'*  have  found  a  marked  seasonal  variation 
in  the  thyroid  iodin  of  animals,  there  being  about  three  times  as  much 
between  June  and  November  as  between  December  and  May.'*' 
In  man  it  has  been  found  that  before  birth  the  thyroid  of  the  fetus 
contains  little  or  no  iodin,  but  in  domestic  animals  the  fetal  glands  con- 
tain not  a  little  iodin  (Fenger).*^  The  cells  of  the  gland  contain  very 
little  iodin  (A.  Kocher).  Extracts  of  the  thyroid  have  little  effect 
on  the  blood  pressure,  except  for  an  alcohol-soluble  fraction,  poor  in 
iodin,  which  is  a  depressor. "^^  On  the  other  hand,  thyroid  secretion 
increases  the  sensitiveness  of  the  sympathetic  nervous  system  to 
epinephrine.^^ 

Wasting  diseases  are  associated  with  a  considerable  decrease  in  the 
size  of  the  thyroid  and  the  amount  of  colloid,  and  with  this  a  decrease 

^"Fonio,  Mitt.  Grenz.  Med.  u.  Chir.,  1911  (24),  123;  Frey,  ibid.,  1914  (28), 
349;  Hunt,  Jour.  Amer.  Med.  Assoc,  1907  (49),  1323;  and  Jour.  Pharm.  and  exp. 
Therap.,  1910  (2),  15. 

*'  Marine  and  Rogoff,  Jour.  Pharm.,  1916  (9),  1. 

*-  Arch.  Int.  Med.,  1909  (4),  440. 

*3  Jour.  Amer.  Med.  Assoc.  1897  (29),  897. 

"  Jour.  Biol.  Chem.,  1913  (13).,  517. 

■*=  Valuable  figures  on  the  iodin  content  of  foods  are  given  bj-  Forbes  et  al., 
Bullet.  Ohio  Agric  Expt.  Station,  June,  1916  No.  299. 

«  Jour.  Biol.  Chem.,  1912  (11),  489;  1912  (12),  55;  1913  (14),  397. 

"^  Fawcett  et  al.,  Amer.  Jour.  Physiol.,  1915  (36),  113. 

'^^  The  thyroid  is  very  rich  in  lipase,  catalase  and  peroxidase;  extirpation  is 
followed  by  a  decrease  in  these  enzymes  in  the  blood,  while  thyroid  feeding  in- 
creases them  as  well  as  the  antitrypsin  (Juschtschenko,  Biochem.  Zeit.,  1910  (25), 
49;  Zeit.  physiol.  Chem.,  1911  (75),  141.). 


604         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

in  the  iodin;  especially  is  this  true  of  tuberculosis/^  Patients  or  ani- 
mals to  whom  iodin  compounds  are  administered  deposit  it  in  the  thy- 
roid in  large  amounts,  especially  if  the  gland  is  previously  defective 
in  iodin,  and  at  times  there  results  even  an  acute  thjToiditis  from  the 
iodin  administration.^"  Iodides  are  said  to  increase  the  amount  of 
thyreoglobulin  itself  ( Wiener). ^^  The  variation  in  iodin  content 
under  various  conditions  is  given  in  the  following  table  from  Jolin,^^ 
his  figures  for  normal  glands  being  somewhat  lower  than  found  in 
America. 


Number  and  condition  of  glands 


Dry  wt. 
gnis. 


Mg.  iodin         Total 
per  gm.  iodin 


r  I  \  152  glands  from  persons  over  10  yrs.  old  (44  notl 

/«                  normal) i  7 .  04 

.y    **  «■          28  glands  from  children  (1  mon.  to  10  yrs.j 0.54 

/k               108  normal  glands  from  adults  only  (both  sexesj ....  5 .  38 

/     ^  «.             67  normal  glands  from  adults  (males) 5 .  07 

41  normal  glands  from  adults  (females) 5.90 

38  glands  from  chronic  diseases 4.29 

29  glands  from  acute  diseases 5.54 

21  glands  from  sudden  death 6 .88 

10  glands  showing  marked  goiter 23.09 

25  colloid-rich  glands ;  8.25 

34  glands  from  persons  receiving  iodin 5 .  79 


1.63 

11.20 

0.28 

0.145 

1.56 

8.05 

1.56 

7.83 

1.55 

8.40 

1.90 

7.81 

1.47 

8.11 

1.29 

8.45 

1.09 

26.49 

2.24 

18.20 

2.56 

15.06 

Chemistry  of  Goiter 
In  connection  with  his  earliest  studies  of  thjToiodin  Baumann  ob- 
served a  great  difference  in  the  amount  of  iodin  in  the  thyroid  glands 
of  normal  individuals  living  in  goitrous  districts,  as  compared  with 
those  living  in  non-goitrous  districts.  Thus  in  Freiburg,  a  goitrous 
district,  the  average  weight  of  the  dried  thyroid  was  8.2  grams,  each 
gram  containing  0.33  mg.  of  iodin,  a  total  of  2.5  mg.  of  iodin  to  each 
gland.  Glands  from  Hamburg  averaged  4.6  gm.  in  weight,  containing 
0.83  mg.  of  iodin  per  gram,  a  total  of  3.83  mg.  per  gland.  Berlin 
glands  weighed  7.4  grams,  and  contained  0.9  mg.  of  iodin  per  gram, 
or  a  total  of  6.6  mg.  of  iodin  per  gland.  Both  of  the  last-named  cities 
are  in  districts  where  goiter  is  not  endemic.  The  thyroids  of  young 
children  show  the  same  relative  paucity  of  iodin  in  goitrous  districts, 
as  compared  with  non-goitrous  districts.  Wells''^  found  that  the  thy- 
roids throughout  the  United  States  contain  even  larger  amounts  of 
iodin  than  the  Berlin  glands,  averaging  10  to  12  mg.  per  gland,  agree- 
ing with  the  fact  that  goiter  is  comparatively  rare  in  this  country.'''* 

"  See  Vitrey  and  Giraud,  Compt.  Rend.  Soc.  Biol.,  1908  (65),  405.  Aesch- 
bacher,  Mitt.  Grenz.  Med.  Chir.,  1905  (15),  269;  Pellagrini,  Arch.  sci.  Med.,  1915 
(39),  276. 

^0  See  Mendel,  Med.  Klinik,  1906  (2),  833. 

"Arch.  e.xp.  Path.  u.  Pliarm.,  1909  (61),  297. 

^'^  Festschr.  f.  O.  Ilammarstcn,  Upsala  I.akarcforen.  Forh.,  1906,  XI,  Suppl. 

"  Jour.  Amcr.  Med.  Assoc,  1897  (29),  897. 

"  It  is  probable,  in  view  of  the  higher  results  obtained  by  later  analyses,  that 
the  results  of  Baumann  and  of  Monerv  are  somewhat  too  low. 


CHEMISTRY  OF  dOITER  fiOo 

Moncry"  has  found  for  France,  as  Bauniann  did  for  (jcrmany,  liiat 
the  amount  of  iodin  contained  in  the  ghmds  of  normal  incUviduals  is 
in  inverse  proportion  to  the  frcqiioncy  of  p;oitpr  in  districts  in  which 
they  Hvc.  Oswakl,  and  also  Acschbachcr,''''  however,  slate  that  normal 
thyroids  in  goi^i'O'^is  districts  contain  inoie  io(Un  than  thyroids  from 
goiter-free  districts. 

Chemical  analyses  of  goiters  have  given  extremely  variable  results, 
which  arc  found  to  depend  upon  the  histological  type  of  the  goiter. 
Baumann  found  that  in  a  series  of  twelve  cases  of  goiter,  in  which  the 
average  dry  weight  was  32  grams,  the  amount  of  iodin  in  each  gram 
was  but  0.09  mg.,  but  the  total  amount,  2.6  mg.,  was  about  the  same 
as  in  normal  glands  of  the  same  goitrous  district.  However,  in  two 
goiters  large  amounts  of  iodin  were  found,  nameh',  17.5  n'g.  and  31.5 
nig.  Wells  found  that  the  amount  of  iodin  depended  upon  the  struc- 
ture, for  two  hyperplastic  goiters  contained  respectively  8.23  and  8.3 
mg.  of  iodin,  or  about  the  amount  normal  for  thyroids  in  this  country, 
whereas  two  colloid  goiters  contained  53.16  and  24.59  mg.  of  iodin. 
This  is  corroborated  by  the  more  extensive  studies  of  Marine  and  his 
co-workers,  who  have  found  the  proportion  of  iodin  low  in  all  glands 
showing  epithelial  hyperplasia,  but  high  in  colloid  goiters. ^'^  Admin- 
istration of  iodin  causes  a  reversion  of  the  hyperplastic  to  the  colloid 
type  of  gland,  while  deprivation  of  iodin  causes  hyperplasia.  Pre- 
sumably, therefore,  during  the  active  growth  of  a  goiter  the  iodin 
is  low,  but  in  the  quiescent  colloid  state  it  is  high.  The  physiological 
activity  of  colloid  or  other  preparations  from  goiters  is  found  to  be 
quite  the  same  as  that  from  normal  glands,  varying  in  direct  proportion 
to  the  iodin  content. ^^  In  an  adenomatous  goiter,  in  the  new  growth, 
Wells  found  1.98  mg.  of  iodin  per  gram,  while  the  rest  of  the  gland  con- 
tained but  0.8  mg. ;  the  total  amount  of  iodin  was  9.26  mg.,  or  the  same 
quantity  as  found  in  normal  glands.  In  nine  fetal  adenomas  Marine 
and  Lenhart  found  iodin  in  eight  in  amounts  averaging  0.174  mg.  per 
gram  of  dry  weight.  However,  when  iodin  is  given  to  persons  with 
thyroid  adenomas  the  tumor  tissue  does  not  take  up  the  iodin  to  the 
same  extent  that  the  normal  gland  tissue  does.  The  presence  of 
great  numbers  of  mitochondria  in  the  cells  of  adenomas  indicates  their 
high  functional  activity. -^^ 

Oswald  found  that  colloid  goiters  contain  a  thyreoglobulin  that  is 
relatively  very  poor  in  iodin;  in  goiterous  calves  the  thyreoglobulin 
contained^no  iodin;  in  human  goiters  it  contained  but  0.07  to  0.19 
per  cent,  of  iodin,  as  against  a  normal  proportion  of  0.34  per  cent. 
Administration  of  iodides  to  a  goiterous  patient  caused  a  rise  in  the 
proportion  of  iodin  in  the  colloid  to  0.51  per  cent.,  showing  that  in 

»  Jour.  Pharm.  et  Chim.,  1904  (95),  288. 

^"  Mitt.  a.  d.  Grenzgeb.  Med.  u.  Chir.,  1905  (15),  269. 

"  Arch.  Int.  Med.,  1908  (1),  349;  1909  (3),  CO;  1909  (4),  440. 

58  See  Fonio,  Mito.  (irenz.  Med.  u.  Chir.,  1911  (24),  123. 

"  Goetsch,  N.  Y  State  Jour.  Med.,  July,  1918. 


606         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

colloid  goiters  in  goitrous  districts  the  thyreoglobulin  is  probably  poor 
in  iodin  because  of  a  lack  of  iodin  for  it  to  unite  with,  and  not  because 
it  is  of  an  abnormal  nature  that  prevents  its  chemical  combination 
with  iodin.*'"  Possibly  this  explains  the  greater  iodin  content  observed 
in  colloid  goiters  in  the  United  States  as  compared  with  colloid  goiters 
observed  in  goitrous  districts.  In  general,  Oswald"  found  the  amount 
of  iodin  to  vary  with  the  amount  of  colloid  in  the  goiters,  although 
occasionally  goiters  with  exceptionally  large  amounts  of  iodin  were 
found,  and  the  proportion  of  iodin  is  not  usually  so  great  when  the 
amount  of  colloid  is  very  large.  Simple  hyperplastic  goiters  he  found 
poor  in  iodin,  or  free  from  it  if  they  contained  no  colloid;  however, 
they  were  found  to  contain  a  thyreoglobulin  typical  in  all  respects 
except  an  absence  of  iodin.  Presumably  in  such  goiters  the  little 
thyroiodin  present  is  contained  in  the  parenchymatous  cells.  The 
physiological  activity  of  thyreoglobulin  obtained  from  goiters  was 
found  to  be  the  same  as  that  from  normal  glands,  except  that  it  was 
weaker  in  direct  proportion  to  the  amount  of  iodin  it  contained,  and, 
therefore,  when  iodin-free  it  was  without  effect.*'-  In  colloid  goiters 
the  greater  part  of  the  weight  of  the  gland,  three-fourths  or  more,  is 
made  up  of  this  colloid-poor  thyreoglobuKn.  The  fluid  contents  of 
cystic  goiters  may  be  free  from  iodin,  but  if  they  contain  much  colloid, 
iodin  will  be  found,  and  Rositzky*''^  found  0.193  mg.  of  iodin  in  20  c.c. 
of  the  jelly-hke  contents  of  a  thyroid  cyst. 

It  has  been  frequently  suggested  that  the  cause  of  endemic  goiter 
is  a  deficiency  in  the  iodin  in  the  food,  or  in  the  drinking-water,  or  in 
the  air  of  the  goitrous  district.  This  is  supported  by  the  relative  in- 
frequency  of  endemic  goiter  in  districts  on  the  sea-coasts,  where  the 
iodin-containing  sea-water  is  sprayed  through  the  air,  and  where  the 
inhabitants  eat  largely  of  sea-foods.  Also  administration  of  minute 
amounts  of  iodin,  even  in  the  air,  seems  to  reduce  existing  goiter  and  to 
prevent  its  occurrence  in  inhabitants  of  goitrous  districts.''"'  However, 
there  are  many  exceptions,  and  it  cannot  be  said  that  this  hypothesis 
of  the  etiology  of  all  goiter  rests  on  satisfactory  evidence,  particularly 
in  view  of  the  abundant  iodin  content  of  colloid  goiters.  Epidemics 
of  goiter  presumably  are  the  results  of  an  infection  with  some  unknown 
organism,  and  possibly  the  endemic  form  has  a  similar  cause. "^  -  There 
is  much  evidence,  in  any  event,  that  whatever  the  cause  of  goiter  may 
be,  it  often  is  related  to  the  drinking  water,""  but  numerous  well-con- 
trolled experiments  fail  to  support  this  hypothesis.""     Verj^  probably 

8"  See  Kocher,  Mitt.  a.  d.  Grenzgeb.  Med.  u.  Chir.,  1905,  vol.  14. 

"  Virchow's  Arch.,  1902  (169),  444. 

"  See  Oswald,  Arch.  ges.  Physiol.,  1916  (164),  506. 

"  Wein.  klin.  Woch.,  1897  (10),  823. 

«••  See  Hunzikcr,  Corr.  Bl.  Schw.  Aerzte,  1918  (48),  220. 

"  See  McCarrison,  "The  Thyroid  Gland,"  New  York,  1917. 

"8  See  de  (^uervain,  Mitt.  a.  d"  Grenzgeb.  Med.  u.  Chir.,  1905  (15),  297;  Birchcr, 
Zeit.  exp.  Path.  u.  Ther.,  1911  (9),  1. 

"  See  Munch,  med.  Woch.,  1913  (60),  393  and  1813;  Sitzber.  Wien.  Akad.,  1914 
(123),  35. 


MYXEDEMA  AND  CRETINISM  (i07 

the  causes  of  colloid  goiter  and  paieiicliymatous  goiter  will  be  found 
to  be  different  from  the  causes  of  cystic  and  adenomatous  goiters. 

Myxedema  and  Cretinism 

These  conditions  depend  upon  a  deficiency  of  thyroid  secretion, 
whether  from  operative  procedure  or  from  pathological  alterations  in 
the  organ.  Consequently  we  find  evidences  of  a  decreased  protein 
metabolism,  the  urine  containing  a  diminislKMl  quantity  of  nitrogen, 
especially  in  the  form  of  urea,  while  ammonia  and  other  forms  of 
nitrogen  are  relatively  excessive.  A  retention  of  nitrogen  and  phos- 
phorus has  been  found,  but  not  of  calcium  and  chlorine.'^'*  The 
temperature  is  usually  subnormal,  and  thcenergy  metabolism  is  low. ^* 
Basal  metabolism  is  lower  than  in  any  other  known  condition  (Du 
Bois).'^°  Fat  and  carbohj-drate  metabolism  seem  not  to  be  propor- 
tionately affected, ^^  and  hence  the  elimination  of  CO2  is  relatively  high 
as  compared  to  the  nitrogen  elimination. ,  Gastro-intestinal  disturb- 
ances are  common,  with  resulting  increase  in  the  amount  of  indican 
and  ethereal  sulphates  in  the  urine.  Whether  from  this  cause  or 
from  deep-seated  metabolic  anomalies,  there  is  a  decided  anemia,  and 
the  ability  of  the  corpuscles  to  combine  with  oxygen  seems  to  be 
decreased,  so  that  the  arterial  blood  may  contain  less  oxygen  than 
normal  venous  blood.  It  is  impossible  to  say  whether  the  failure  of 
growth  and  development  of  the  young  (cretinism),  and  the  mental 
and  physical  torpidity  of  the  adult,  are  due  to  an  autointoxication  from 
products  of  intermediary  metabolism  which  accumulate  because  of  the 
failure  of  the  thyroid  to  furnish  the  "stimulus"  necessary  for  their 
complete  destruction,  or  to  a  lack  of  some  essential  action  of  the  thy- 
roid secretion  upon  the  nervous  tissues  and  the  growing  cells  them- 
selves. Administration  of  thyroid  extract  to  cretinoid  children  causes 
retention  of  nitrogen  and  phosphorus,  but  more  strikingly  of  cal- 
cium,"- and  obese  cretins  lose  weight,  chiefly  from  the  non-nitrogenous 
elements  (Scholz).  The  amount  of  iodin  in  human  cretin  thyroids 
seems  not  to  have  been  estimated,  but  in  five  cretin  dogs  Marine  and 
Lenhart  could  find  no  thyroid  iodin  at  all." 

The  myxedematous  change  in  the  connective  tissues  is  in  the  nature 
of  a  reversion  to  the  fetal  type  of  tissue,  and  suggests  that  the  thyroid 

68  Benjamin  and  Reuss,  Jahrb.  f.  Kinderheilk.,  1908  (67),  261.  In  a  cretin 
Greenwald  found  little  deviation  from  normal.  (Arch.  Int.  Med.,  1914  (14), 
374.) 

«9  Talbot,  Amer.  Jour.  Dis.  Chil.,  1916  (12),  145. 

"0  Arch.  Int.  Med.,  1916  (17),  915;  Means  and  Aub.,  ibid.,  1919  (24),  404. 

"  Rarely  myxedema  and  diabetes  have  been  observed  conjointly  (see  Strasser, 
Jour.  Amer.  Med.  Assoc,  1906  (44),  765). 

"  See  Hougardy  and  Langstein,  Zeit.  f.  Kinderheilk.,  1905  (61),  633.  Full 
figures  are  given  by  Scholz,  Zeit.  exp.  Path.  u.  Thcr.,  1906  (2),  270. 

"  Related  to  cretinism  is  the  "hairless  pig  malady,"  in  which  hairless  pigs  are 
born  dead  or  die  soon  after  birth,  the  mothers  and  offspring  often  having  goiter; 
administration  of  KI  checks  the  appearance  of  the  disease  (See  Hart  and^Steen- 
bock.  Jour.  Biol.  Chem.,.1918  (33),  313). 


608 


CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 


secretion  is  necessary  for  proper  cell  growth.  This  effect  might  be 
either  specific,  or  depend  simply  on  the  effect  on  protein  metabolism. 
Horsley^"*  describes  the  appearance  of  the  tissues  of  animals  dying 
after  thyroidectomy  as  follows:  "The  subcutaneous  connective  tissue 
is  swollen,  jelly-like,  bright  and  shining,  and  excessively  sticky.  The 
same  thing  is  observed  in  the  loose  tissue  of  the  mediastinum,  about 
the  heart,  and  in  the  omentum.  The  submaxillary  and  parotid  glands 
are  greatly  enlarged,  and  have  a  semi-translucent,  swollen  appear- 
ance; from  the  cut  surface  a  sticky,  glairy  fluid  exudes.  Apparently 
the  parotid  becomes  transformed  into  a  mucous  gland;  likewise  the  mu- 
cous membrane  of  the  alimentary  tract  is  swollen  and  transparent." 
Fetal  tissues  contain  normally  more  mucin  than  those  of  adults  (0.76 
per  cent,  as  against  0.37  per  cent,  in  the  subcutaneous  tissues,  accord- 
ing to  Halliburton),  and  in  the  early  stages  of  the  formation  of  ex- 
cessive subcutaneous  tissue  in  myxedema  such  an  increase  of  mucin 
may  be  present.  But,  under  ordinary  conditions,  the  term  myxedema 
seems  to  be  entirely  a  misnomer,  for  Halliburton's  analyses  showed 
that  the  skin  of  myxedematous  patients  contains  quite  the  same 
amount  of  mucin  as  is  present  in  normal  skin.'^^  When  the  condition  is 
of  long  standing,  the  amount  of  mucin  may  even  be  much  reduced,  be- 
cause of  the  development  of  a  fibroid  character  in  the  connective  tissue. 
However,  in  monkeys  upon  which  thyroidectomy  had  been  performed, 
Halliburton^^  found  a  decided  increase  in  the  mucin  in  the  tissues 
throughout  the  body,  especially  in  the  salivary  glands,  but  also  in  the 
skin,  subcutaneous  tissues,  and  tendons;  and  mucin  was  found  in  the 
blood,  as  shown  by  the  following  table: 


Skin 

and 

sub- 

aneous 

tissue 


Tendon 


Muscle 


Parotid 


Sub- 
max- 
illary 


Blood 


Normal  monkey 

Normal  monkey . .  .^.  .  . 
After  thyroidectomy — 

55  days 

33  days 

49  days 

7  days 


0.89 
0.9 

3.12 

2'3' 
0.45 


0.39 
0,5 

2.55 

2.4  ' 
0.904 


trace 
0 


0.72 

1^7' 
trace 


0.1 

6.0 

3^3' 
0.16 


0 
0 

0.35 

trace 
0.8 

trace 


It  has  been  suggested  that  the  thyroid  produces  an  enzyme  which 
destroys  mucin,  but  that  such  is  the  case  has  never  been  demon- 

"  Brit.  Med.  Jour.,  1885  (i),  211. 

"  Jour,  of  Pathol,  and  Bact.,  1893  (1),  90. 

'*  (Quoted  l)y  llorsley,  loc.  cit.  Later  e.\pcrimcnters,  however,  have  had  diffi- 
culty in  producing  experimental  my.xedema  as  described  by  Horsley,  or  have 
failed  entirely. 


EXOPHTHALMIC  GOITER  609 

strated."     Levin"  states   tliat   mucin   is  toxic  for  thyroidcctomized 
rabbits,  but  this  is  not  substantiated  by  N6f<5diefT." 

That  the  thyroid  is  connected  witli  general  growth  is  shown  not 
only  by  the  thyroid  abnormalities  present  in  cretinism,  but  also  by 
the  frequent  observation  of  thyroid  defects  in  conditions  of  delayed 
growth  and  development  of  less  extreme  degree  {infantilism) ,  and  the 
favorable  effects  of  thyroid  feeding  in  many  such  cases.  Also  in  cer- 
tain types  of  short-limbed  dwarfs  (chondrodystrophia  Jodnlis)  some 
thyroid  anomaly  maj^  have  an  etiologic  bearing,  for  in  such  a  case,  in 
which  the  thyroid  was  histologically  greatly  altered  and  quite  free 
from  colloid,  I  could  find  no  trace  of  iodin.*"  On  the  other  hand,  the 
thyroid  of  a  giant  which  I  have  analyzed  contained  62.9  mg.  of  iodin, 
or  six  times  the  amount  present  in  normal  glands.*' 

Exophthalmic  Goiter 

It  has  by  no  means  been  conclusively  determined  that  exophthalmic 
goiter  is  due  to  an  intoxication  with  excessive  amounts  of  thyroid  se- 
cretion, either  normal  or  abnormal,  but  there  is  abundant  evidence  in 
favor  of  this  view.  Most  important  is  the  similarity  of  exophthalmic 
goiter  to  the  effects  of  "hyperthyroidism"  or  "thyroidismus,"  pro- 
duced either  experimentally  or  through  overuse  of  thyroid  extract  for 
therapeutic  purposes.  In  thyroidismus  there  are  observed  a  rapid, 
weak  pulse;  greatly  increased  metabolism,  especially  of  proteins ;^2 
a  strildng  increase  in  basal  metabolism,  paralleling  the  degree  of 
intoxication;  marked  mineral  loss,  especially  of  Ca  and  P  from  the 
bones ;^^  increased  secretion,  especially  of  perspiration;  marked  ner- 
vousness and  irritability,  often  with  mental  confusion  and  delusions; 
gastro-intestinal  disturbances,  especially  diarrhea;  sweating,  flushing, 
tremors,  palpitation  of  the  heart,  loss  of  weight,  and  slightly  increased 
temperature  are  also  often  observed,  and  not  rarely  typical  exoph- 
thalmos may  appear.*'*  These  manifestations,  which  are  common  to 
both  thyroidism  and  to  exophthalmic  goiter,  are  quite  the  opposite  of 
the  characteristic  changes  of  myxedema,  w4th  its  general  lowering  of 
all  metabolic  and  nervous  processes.  Alike  in  experiniental  hyperth}-- 
roidism  and  exophthalmic  goiter  there  is  a  greatly  increased  sensitive- 
ness of  the  sympathetic  nervous  system  to  epinephrine.**     Reid  Hunt's 

"  See  Parhon,  Compt.  Rend.  Soc.  Biol.,  1916  (79),  504. 

^8  Med.  Record,  1900  (57),  184. 

"  Vratch,  1901  (22),  Oct.  27. 

80  Reported  bv  Hektoen,  Amer.  Jour.  Med.  Sci.,  1903  (125),  751. 

«i  Reported  by  Bassoe,  Trans.  Chicago  Path.  Soc,  1903  (5),  231. 

^-  Metabolism  in  exophthalmic  goiter,  see  Du  Bois,  Arch.  Int.  Med.,  1916  (17), 
915:  Halverson,  Bergeim  and  Hawk,  ibid.,  1916  (18),  800;  Meaas  and  Aub,  Arch. 
Int.  Med.,  1919  (24),  645. 

83  Kummer,  Rev.  M6d.  Suisse  Rom.,  1917  (55),  442. 

*^  Sugar  utilization  is  decreased,  as  shown  by  study  of  the  utilization  of 
sugar  given  intravenously  (\Vilder  and  Sansum,  Arch.  Int.  Med.,  1917  (19),  311) : 
also  a  dietary  hvperglucemia  is  readily  induced  (Denis,  Aub  and  Minot.  Arch. 
Int.  Med.,  1917  (20),  964;  McCaskv,  Jour.  Amer.  Med.  Assoc,   1919  (73),  243). 

85  See  Goettsch,  N.  Y.  State  Jour.  Med.,  July,  1918. 

39 


610         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

acetonitrile  test  for  thyroid  secretion  has  been  found  positive  in  the 
blood  from  patients  with  exophthalmic  goiter, ^^  which  presumably 
means  the  presence  of  an  excess  of  thyroid  secretion  circulating  in  the 
blood  in  this  disease.  Furthermore,  the  histological  changes  observed 
in  the  thyroid  may  resemble  those  of  compensatory  hypertrophy, 
suggesting  strongly  that  the  goitrous  change  of  this  disease  is  due  to  a 
true  hypertrophy,  with  increased  production  of  the  specific  secretions. 
There  is  a  marked  increase  in  the  mitochondria  of  the  thyroid  epithe- 
lium in  exophthalmic  goiter,  which  also  is  evidence  of  heightened  activ- 
ity.^'' Kocher^^  says  that  when  iodin  is  given  to  patients  with  cancer 
of  the  thyroid  they  may  develop  symptoms  of  exophthalmic  goiter,  as 
if  an  excess  of  thyroiodin  were  formed.  Also  speaking  strongly  in 
favor  of  the  view  that  exophthalmic  goiter  is  the  result  of  overactivity 
of  the  thyroid,  is  the  frequent  cure  of  the  disease  through  removal  of  a 
large  part  of  the  diseased  gland.  Although  at  times  the  colloid  type 
of  gland  is  found  in  exophthalmic  goiter,  Marine  contends  that  it  has 
been  preceded  by  a  hyperplastic  stage. ^^ 

Oswald^°  found  that  the  thyroid  in  exophthalmic  goiter  contains 
generally  a  smaller  proportion  of  iodin  than  normal  glands,  but  with 
the  total  amount  approximately  normal.  However,  the  findings  are 
very  inconstant,  corresponding  with  the  fact  that  in  some  cases  of 
exophthalmic  goiter  the  amount  of  colloid  is  abundant  (in  which  case 
the  amount  of  iodin  may  be  large),  while  usually  the  amount  of  colloid 
is  small,  and  its  highly  vacuolated  condition  in  hardened  sections 
suggests  that  it  is  of  unusually  fluid  consistency.  A.  Kocher^^  found 
that  either  the  amount  of  iodin  is  small,  which  is  usual,  or  else  very 
high,  but  it  is  seldom  the  same  as  in  normal  thyroids;  the  more  dense 
the  colloid  in  the  follicles  the  higher  iodin  content  he  observed;  the 
phosphorus  content  is  both  relatively  and  absolutely  increased.  Ma- 
rine has  found  that  in  exophthalmic  goiter  as  well  as  in  other  conditions 
the  amount  of  iodin  is  in  direct  proportion  to  the  colloid  and  inverse 
to  the  hyperplasia.  E.  V.  Smith^-  obtained  in  simple  hyperplastic 
glands  an  average  of  0.54  mg.  of  iodin  per  gram  dry  weight,  as  com- 
pared with  1.52  mg.  in  hyperplastic  glands  showing  retrogressive 
changes  with  more  densely  staining  colloid.  Fonio  found  that,  as  with 
normal  thyroids,  the  physiological  effect  of  exophthalmic  goiter  glands 
varies  directly  with  the  proportion  of  iodin,  and  such  glands  take  up 
iodin  administered  therapeutically  just  as  a  normal  thyroid  does 
(Kocher,'-*^  Marine  and  Lenhart).^'*     These  results,  therefore,  indicate 

'  8"  See  Ghedini,  Wien.  klin.  Woch.,  1911  (24),  736;  Hunt  and  Seidell,  Jour. 
Pharm.  and  Exp.  Ther.,  1910  (2),  15. 

8'  Goetsch,  Bull.  .lohns  Hopkins  Hosp.,  1910  (27),  129. 

88  Deut.  Zeit.  Chir.,  1908  (91),  302. 

8»  See  also  Wilson,  Ainer.  .Jour.  Med.  Sci.,  1908  (136),  851. 

"o  Virchow's  Arch.,  1902  (169),  475. 

"i  Virchow's  Arch.,  1912  (208),  86. 

»2  Jour.  Amer.  Med.  Assoc,  1914  (62),  113. 

»3  Arcli.  klin.  Chir.,  1910  (92),  442;  1911  (96),  403. 

"  Arch.  Int.  Med.,  1911  (8),  265. 


EXOl'UTllALMIC  GOITER  (ill 

nothing  cither  for  or  ajrainst  the  hypothesis  that  cxoi)hthalmic  goiter 
is  due  to  autointoxication  with  the  secretion  of  the  tliyroid,  but  Wilson 
and  KendalP-^  find  that  in  the  toxic  type  of  goiters  there  is  but  Ho- 
}i5  as  much  of  the  active  iodin  compound  of  Kendall  as  in  normal 
glands,  and  hence  they  suggest  that  in  thyroid  intoxication  this  toxic 
material  has  been  discharged  from  the  thyroid  into  the  circulation. 

On  the  other  hand,  it  is  impossible  to  produce  a  symptom-complex 
completely  resembhng  exophthalmic  goiter'-"'  in  animals  by  excessive 
feeding  of  thyroid,"  either  normal  or  from  exophthalmic  goiter;  and 
after  extensive  study  of  the  subject  Marine  and  Lenharthave  come  to 
the  conclusion  that  "the  essential  phj'siological  disturbance  of  the 
thyroid  in  exophthalmic  goiter  is  insufficiency,  its  reaction  com- 
pensatory and  its  significance  symptomatic."  This  view,  however, 
certainly  fails  to  agree  with  the  excellent  results  which  come  from 
partial  extirpation  of  the  thyroid  in  exophthalmic  goiter.  Oswald, ^^ 
also  an  experienced  investigator  in  this  field,  invokes  an  abnormally 
irritable  nervous  system,  which  stimulates  the  thyroid  and  in  turn  is 
stimulated  by  the  thyroid  secretion,  constituting  a  vicious  circle. 
Other  observers  are  of  the  opinion  that  not  an  excessive,  but  a  per- 
verted, secretion  is  at  fault, ^'  a  view  not  confirmed  by  tests  of  the 
effects  of  thyroid  extracts  on  animals.^  However,  it  is  stated  by 
Blackford  and  Sanford,^  that  extracts  of  the  thyroid  in  this  disease,  as 
well  as  the  blood  of  patients  in  the  acute  toxic  stages,  exhibit  a  marked 
depressor  effect  on  blood  pressure,  which  is  distinct  from  that  of 
chohne,  and  which  they  believe  to  be  specific  for  exophthalmic  goiter. 

There  can  be  no  doubt  that  the  thyroid  secretion  is  capable  of  caus- 
ing serious  intoxication,  for  patients  who  have  overused  thyroid  prep- 
arations in  the  treatment  of  obesity,  skin  diseases,  etc.,  have  often 
suffered  severely  from  the  symptoms  mentioned  previously,  and,  in  at 
least  one  such  case,  a  diagnosis  of  exophthalmic  goiter  was  made  be- 
fore the  cause  of  the  disturbance  was  detected.  Not  infrequently 
evidences  of  acute  intoxication  have  followed  immediately  after 
operations  upon  the  thyroid,  and  these  have  been  considered  as  due  to 
intoxication  with  the  large  quantities  of  thyroid  secretion  that  have 
escaped  from  the  gland  during  the  operative  manipulation.  The  fact 
that  amhlyopia,  resembling  that  produced  by  tobacco,  etc.,  may  follow 
overuse  of  thyroid  preparations^  is  indicative  of  their  toxicity',  as 
also  is  the  glycosuria  that  may  result  from  thyroid  administration.^ 

95  Amer.  Jour.  Med.  Sci.,  1916  (151),  79. 

9^  The  pathogenesis  of  the  exophthalmos  is  unknown.  See  Troell,  Arch.  Int. 
Med.,  1916  (17),  382. 

"  See  Carlson  et  al,  Amer.  Jour.  Physiol.,  1912  (30),  129;  Marine,  Jour.  Amer. 
Med.  Assoc,  1912  (59),  325. 

5*  Correspondenzblatt  Schweizer  Aerzte,  1912  (42),  1130. 

99  Klose  et  al,  Beitr.  z.  klin.  Chir.,  1912  (77),  601. 

1  See  Schonborn,  Arch.  exp.  Path.  u.  Pharm.,  1909  (60),  390. 

2  Jour.  .\mer.  Med.  Assoc,  1914  (62),  117. 

3  Birch-Hirschfeld  and  Inouve,  Graefe's  Arch.,  1905  (61),  499. 
^See  Geyelin,  .\rch.  Int.  Med.,  1915  (16),  975. 


612         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

Even  if  the  hypothesis  that  exophthalmic  goiter  is  due  to  intoxi- 
cation with  thyroid  secretion  is  correct,  we  have  no  satisfactory  ex- 
planation of  the  cause  of  the  hyperactivity  of  the  thyroid.  In  some 
cases  degenerative  changes  have  been  observed  in  the  superior  cervical 
sympathetic  ganglia,  and  cure  or  improvement  of  exophthalmic  goiter 
is  said  to  have  followed  resection  of  these  ganglia;  however,  this  re- 
lation has  not  been  observed  at  all  constantly.  In  other  cases  there 
has  been  evidence  that  suggested  a  primary  intoxication  with  the  prod- 
ucts of  intestinal  putrefaction,  leading  to  a  secondar}^  hyperplasia  of 
the  thyroid,  but  this  also  seems  to  be  an  exceptional  observation. ° 
All  things  considered,  it  seems  most  probable  that  the  h3'^peractivity 
of  the  thyroid  is  due  to  some  exciting  condition,  and  is  not  of  itself 
primary,  although  the  resulting  hypersecretion  of  the  thyroid  may 
cause  the  dominant  features  of  the  disease.  The  frequent  association 
of  exophthalmic  goiter  with  puberty  and  pregnancy  suggests  that 
some  abnormality  in  the  function  of  the  generative  organs  may  be  a 
frequent  starting-point  of  the  thyroid  derangement. "^  In  not  a  few 
cases  diabetes  or  pancreatitis  have  been  associated,^  and  some 
observers  state  that  the  pressor  substance  (presumably  epinephrine) 
in  the  blood  is  much  increased  in  exophthalmic  goiter.^  Although 
the  thymus  is  often  found  enlarged,  sometimes  greatly  so,  in  70  to  80 
per  cent,  of  cases  of  exophthalmic  goiter,  its  relation  to  the  disease 
is  as  yet  entirely  unknown.^ 


^  Antithyroid  Serum. — Based  on  the  theory  that  the  normal  function  of  the 
thyroid  is  the  detoxication  of  metabolic  products,  is  the  serum  treatment  advocated 
first  by  Ballet  and  Enriquez,  and  later  by  Lanz,  and  Burghart  and  Rlumenthal. 
(Deut.  med.  Woch.,  1899  (25),  627.  Also  Mohius,  Mlinch.  med.  Woch.,  1901 
(48),  1853;  v.  Leyden,  Med.  Klinik,  1904  (1),  1;  Eulenberg,  Bed.  klin.  Woch., 
i905  (42),  3.)  On  the  principle  that  after  thyroidectomj'  the  blood  should  con- 
tain an  accumulation  ot  those  substances,  which  the  thyroid  normalh'  neutralizes, 
they  injected  the  serum  of  thyroidectomized  goats  into  patients  with  exophthalmic 
goiter,  in  the  hope  that  these  accumulated  substances  might  m  turn  neutralize 
any  excessive  thyroid  secretion.  Favorable  results  were  obtained,  and  it  was 
subsequently  found  that  the  milk  of  thyroidectomized  goats  possesses  the  same 
qualities,  and  may  be  administered  by  mouth;  this  has  led  to  quite  extensive 
clinical  use  of  this  method  of  treatment,  which  has  failed  to  show  anj^  regular 
beneficial  effects  in  the  hands  of  most  careful  observers.  (See  Sonne,  Zeit.  klin. 
Med.,  1914  (80),  229.)  Of  similar  significance  are  the  favorable  etfects  obtained 
bv  Beebe  (Jour.  Amer.  Med.  Assoc,  190a  (40),  484;  1000  (47),  655)  and  Rogers 
(ibid.,  190G  (46),  487;  1906  (47),  661)  with  a  serum  made  by  immunization  of 
animals  with  the  nucleoproteins  of  the  thyroid,  which  have  not  been  corroborated 
by  others. 

*  The  serum  of  patients  with  exophthalmic  goiter  shows  bj"-  Abderhalden's 
method  a  constant  power  to  digest  thyroid  tissue,  and  sometimes  ovarv  or  testicle 
(Lampo  and  Fuchs,  Miinch.  med.  Woch.,  1913  (60),  No.  39). 

^  Thompson,  Amer.  Jour.  Med.  Sci.,  1906  (132),  835;  Colin  and  Peiser,  Deut. 
med.  Woch.,  1912  (38),  60. 

8  Broking  and  Trendelenburg   Deut.  Arch.  klin.  Med.,  1911  (103),  168. 

'  Review  by  Eddy,  Canad.  Med.  Assoc.  Jour.,  March,  1919. 


CHEMISTRY  OF  THE  PARATHYROIDS  til 3 

The  Parathyroids'" 

The  parathyroids  were  originally  considered  as  but  a  form  of  undeveloped 

accessory  thyroids,  but  they  are  now  generally  believed  to  be  independent  organs 
of  fully  as  great  importance  as  the  thyroid.  Tiieir  independence  is  conclusively 
shown  by  tlie  cases  of  cretinoid  ciiildren  in  whom  the  thyroid  proper  has  fiiiled  to 
develop,  while  the  parathyroids  are  found  to  be  normal,"  thus  proving  their 
distinct  origin,  the  iiuibility  of  parath\  roid  tissue  to  change  into  thyroid  tissue,'* 
and  their  inability  to  prevent  the  changes  of  cretinism.'^  Parathyroids  contain 
no  appreciable  amounts  of  iodin  (Estes  and  Cecil),'-'  although  14  per  cent,  of  para- 
thyroids obtained  at  autopsy  contain  a  colloid  material  (Thompson  and  Ilarris)." 
Glycogen  is  demonstrable  in  the  epithelium.  To  their  removal  are  ascribed  by 
many  investigators  the  acute  manifestations  of  athyreosis,  while  tlic  more  clironic 
changes  of  my.xedema  are  attributed  to  the  loss  of  the  thyroid.  MacCJallum's 
studies  support  this  view,  for  he  found  the  results  of  parathyroidectomy  in  dogs 
very  different  from  the  results  of  thyroidectomy.  The  most  prominent  symptoms 
are  muscular  twitchings,  gradual!}'  passing  into  tetanic  spasms,  and  due  to  nervous 
impulse  rather  than  to  muscular  changes,  since  they  did  not  appear  in  muscles 
from  which  the  nerve-supply  has  been  cut  off.  Trismus,  protrusion  of  the  eyes, 
and  rapid  respiration  without  cyanosis  (i.  e.,  air  hunger)  were  al.so  observed,  and 
death  usually  resulted  from  exhaustion.  Apparently  these  symptoms  are  due  to 
some  toxic  substance  which  accumulates  on  account  of  the  absence  of  the  para- 
thjToids,  for  it  was  found  that  simply  diluting  the  dog's  l)lood  by  withdrawing 
part  of  it,  and  injecting  a  corresponding  amount  of  salt  solution,  caused  a  tempor- 
ary cessation  of  the  tetanic  symptoms;  and  injections  of  emulsions  of  parathyroid 
checked  the  symptoms  for  some  time,  presumably  through  neutralizing  the  hypo- 
thetical poisons.  Degenerative  changes  that  were  observed  in  the  cerebral  ganglion- 
cells  also  favor  the  view  that  some  unneutralized  toxin  is  responsible  for  the 
symptoms  following  parathyroidectomy.  On  the  other  hand,  profound  mental 
symptoms  and  insomnia  have  resulted  from  feeding  parathyroid  toman.'"'  Recent 
studies  make  it  seem  probable  that  tetany  parathyreopriva  and  idiopathic  tetany 
are  either  identical  or  very  closely  related.'^ 

The  metabolism  after  parathyroidectomy  may  show  the  following  changes:'* 
There  is  a  reduction  in  the  assimilation  limit  for  carl)ohydrates  (Hirsch,  Under- 
bill'* and  others).  There  is  a  disagreement  concerning  inorganic  metabolism,  for 
while  MacCallum  and  Voegtlin-"  found  an  increased  elimination  of  calcium  and 
a  loss  of  the  same  element  from  the  blood  and  brain  (which  they  would  make 
responsible  for  the  increased  nervous  irritaliility),  Cooke  found  no  such  loss  of 
calcium,-'  but  she  did  find  an  increased  urinary  excretion  of  magnesium.  Ac- 
cording to  most  observers,  nitrogenous  metabolism  is  altered  as  shown  by  the  in- 
creased excretion  of  nitrogen,  and  especially  of  ammonia.  Greenwalci--  found 
increased  ammonia  less  conspicuous  than  increased  undetermined  nitrogen  and 
sulphur,  and  decreased  phosphorus  excretion.  There  maj'  occur  an  increase  in 
the  bases  of  the  blood  (alkalosis,  q.  v.)  which  disappears  under  the  acidosis  that 
results  from  tetany.-^ 

In  view  of  the  conflicting  facts,  the  theory  that  the  increased  irritability  and 

1°  A  review  of  this  subject  is  given  by  Thompson  in  "The  Surgery  and  Pathol- 
ogy of  the  Thyroid  and  Parathyroid  Glands,"  by  A.  J.  Ochsnerand  R.  L.  Thomp- 
son, St.  Louis,  1910.     See  also  MacCallum,  Krgeb.  inn.  Med.,  1913  (11),  569. 

11  Roussv  and  Clunet,  Compt.  Rend.  Soc.  Biol.,  1910  (68),  818. 

12  See  Edmunds,  Jour.  Path,  and  Bact.,  1910  (14),  288. 

13  See  MacCallum,  Johns  Hopkins  Hosp.  Bull..  1907  (18),  341. 

^*  Ibid.,    1907   (18),   331;  also   Cameron,  Jour.  Biol.  Chem.,   1914  (16),   465. 

15  Amer.  Jour.  Med.  Sci.,  1908  (19),  135. 

i«  Morris,  Jour.  Lab.  Clin.  Med.,  1915  (1),  26. 

'^  See  Paton  and  Findlay,  Ouart.  Jour.  Exp.  Physiol.,  1917  (10),  203. 

18  See  review  bv  Cooke,  Amer.    our.  Med.  Sci.,  1910  (140),  404. 

19  Jour.  Biol.  Chem.,  1914  (18),  87. 

20  Jour.  Exp.  Med.,  1909  (11).  118;  1913  (18),  618. 

21  See  also  Bergeim,  Stewart  and  Hawk,  J.nir.  Exp.  Med.,  1914'(20),  225. 
"Amer.  Jour.  Phvsiol.  1911  (28),  103;  Jour.  Biol.  Chem.,  1913  (14),  363. 

"  Wilson,  Stearns  and  Thurlow,  Jour.  Biol.  Chem.,  1915  (23),  89,  123.  Mc- 
Cann,  ibid.,  1918  (35),  553;  Togawa,  Jour.  Lab.  Clin.  Med.,  1920  (5),  299. 


614         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

spasm  of  tetany  result  from  hypocalcification  of  the  nerve  tissue  is  at  present 
unproved.  Calcium  does  diminish  nervous  irritability,  as  shown  by  J.  Loeb, 
and  hence  when  administered  it  may  favorably  influence  the  symptoms  of  tetany 
parathyreopriva,  but  this  does  not  establish  the  theory.  That  numerous  experi- 
menters have  been  able  to  stop  these  symptoms,  both  in  man  and  animals,  by 
feeding  of  parathyroid,^^  or  parathyroid  nucleoprotein,  esiablishes  the  relation- 
ship of  this  gland  to  the  tetany,  but  not  the  calcium  deprivation  hypothesis.  A 
critique  of  this  hypothesis  by  Berkeley  and  Beebe"  brings  out  the  follo\s'ing 
points:  Strontium,  magnesium  and  barium  have  the  same  effect  in  tetany  as 
calcium,  whereas  severe  calcium  loss  in  diabetic  acidosis  does  not  cause  tetany. 
The  fact  that  bleeding  reduces  the  symptoms  is  against  the  calcium  deprivation 
theory  and  supports  the  intoxication  theory.  Wiener^^  even  states  that  it  is 
possible  to  secure  an  antitoxin  for  the  poison  of  tetany  thyreopriva  by  immuniz- 
ing with  the  serum  of  animals  in  tetany.  On  the  other  hand,  the  marked  changes 
in  dentition  and  bone  repair  observed  in  parathyroidectomized  animals  by  Erd- 
heim^''  indicate  an  abnormality  in  calcium  metabolism,  which,|however,  might  be 
secondary  to  an  intoxication.  Also,  in  osteomalacia  and  osteoporosis  the  para- 
thyroids are  said  to  show  hyperplasia,^*  and  Howland  and  Marriott  have  found  a 
definite  decrease  in  the  calcium  of  the  blood  in  human  tetany  and  in  parathyroid- 
ectomized dogs.^^  Injection  of  phosphates  reduces  blood  calcium,  and  when  the 
reduction  has  reached  6  mg.  per  100  c.c,  symptoms  of  tetany  appear.^" 

MacCallum^^  has  found  evidence  that  in  parathyroidectomized  dogs  the  blood 
contains  something  which  greatly  increases  the  irritability  of  the  nerves,  possibly 
by  abstracting  calcium  from  the  tissues.  Removal  of  calcium  from  the  blood  by 
dialysis  results  in  nerve  hyperexcitability  resembling  that  seen  in  tetany.  W.  F. 
Koch^-  found  guanidine  and  methyl  guanidine  in  the  urine  of  dogs  deprived  of 
parathyroids.  Burns  and  Sharpe'^  corroborated  this,  and  also  found  the  same 
bases  in  the  urine  of  children  with  idiopathic  tetany.  Salts  of  guanidine  produce 
typical  symptoms  of  tetany,  including  the  liypoglucemia,  calcium  loss  and  acidosis 
observed  in  this  disease.^*  After  parathyroidectomy  there  is  a  fall  in  the  guanidine 
content  of  the  muscle  (Henderson).'^  Apparently  the  parathyroids  control  the 
metabolism  of  guanidine  in  the  body  by  preventing  its  development  in  undue 
amounts,  in  this  way  exercising  a  regulative  action  on  the  tone  of  the  skeletal  mus- 
cles (Paton  and  Findlay).  Administration  of  guanidine  to  dogs  produces  much 
the  same  symptoms  as  removal  of  the  parathyroids  (Burns). 

Cooke  states  that  the  metabolic  changes  precede,  and  presumably  incite  the 
tetany.  Implantation  of  parathyroid  tissue  in  persons  with  tetany  parathyreo- 
priva has  been  successful  in  removing  symptoms  in  a  few  cases. ^^  The  relation 
of  the  parathyroids  to  tetany  of  infants  is  not  so  well  established,^^  although 
several  observers  have  found  hemorrhages  in  the  parathyroids  in  these  cases. 
Some  cases  of  "gastric  tetany"  have  improved  under  parathyroid  feeding,  which 
is  ;also  said  to  be  beneficial  in  paralysis  agitans,^^  although  there  seems  to  be 
no  anatomic  basis  for  assuming  a  parathyroid  deficiency  in  this  disease. 

The  Relation  of  the  Parathyroids  to  Exophthalmic  Goiter. — This  has  not  yet  been 
definitely  established.  As  nei-vous  manifestations  are  prominent  after  parathy- 
roidectomy, it  has  seemed  probable  that  these  organs  may  Ijo  more  closely 
associated  with  exophthalmic  goiter  than  is  the  thyroid  itself.^'*     Against  the  hypo- 

■'  See  Halsted,  Amer.  Jour.  Med.  Sci.,  1907  (134),  1. 
"Jour.  Med.  Res.,  1909  (20),  149. 
26  Pfliiger's  Arch.,  1910  (136),  107. 
"Frankfurter  Zeit.  Pathol.,  1911  (7),  175. 
28  Todyo,  Frankf.  Zeit.  Pathol.,  1912  (10),  219. 
2»  Trans.  Amer.  Ped.  Soc,  Vol.  2S,  p.  202. 
'0  Binger,  Jour.  Pharmacol.,  1917  (10),  105. 

'1  Verli.  Deut.  Path.  Ges.,  1912  (15),  26(5;  Jour.  Exp.  Med.,  1914  (20),    149. 
32  Jour.  Biol.  Chem.,  1913  (15),  43;  Jour.  Lab.  Clin  Med.,  1916  (1),  299. 
33()uart.  .lour.  Exp.  Phvsiol,  1916  (10),  345. 

^*  See  Watanabe,  Jour.  Biol.  Chem.,  1918  (33),  253;  1918  (36),  531. 
36  Jour.  Pliysiol.,  1918  (51),  1. 
36  Danielsen,  Beit.  klin.  Chir.,  1910  (66),  85. 
3'  See  Haberfeld,  Vircliow's  Arch.,  1911  (203),  282. 
38  Berkeley,  Med.  Record,  191()  (90),  105. 

3»  This  subject  is  thoroughly  reviewed  by  MacCallum,  Med.  News,  1903  (83), 
820;  Iverscn,  Arch.  Internat.  de  Chir.,  1914  (6),  255. 


THE  ADRENALS  AND  ADDISON'S  DISEASE  G15 

thesis  that  exophthalmic  goiter  is  due  to  parathyroid  insufficiency,  however,  stand 
the  following  facts: 

(1)  Removal  of  one  lobe  of  the  thyroid  often  causes  improvement  or  recovery 
in  this  disease,  yet  with  the  lobe  of  the  thyroid  is  generally  removed  the  adjacent 
parathyroid,  which  would  decrease  the  amount  of  i)aralhyroid  tissue,  and  make 
worse  any  existing  parathyroid  insufhciency.  (2)  Therapeutic  administration  of 
parathyroid  tissue  or  extract  has  had  no  significant  effect  on  the  disease.  (3) 
No  considerable  or  characteristic  anatomical  changes  occur  in  the  parathyroids  in 
exophthalmic  goiter,'"  while  the  great  majority  of  all  cases  show  changes  in  the 
thyroid.  (4)  The  parathyroids  seem  to  have  but  slight  influence  on  metabolism 
(MacCallum),  while  metabolic  abnormalities  are  very  marked  in  exophthalmic 
goiter/' 

The  Adrenals  and  Addison's  Disease^- 

Like  the  hypophysis,  the  adrenals  are  essentially  double  organs, 
containing  nervous  and  glandular  tissues.  The  medulla  is  of  sym- 
pathetic nervous  system  origin,  a  part  of  the  chromaffin"  system, 
which  in  most  animals  is  enclosed  in  a  layer  of  entirely  different  nature 
and  origin,  the  cortex  being  an  epithelial  structure,  derived  from  the 
urogenital  anlage,  and  resembling  most  closely  in  structure  (and  per- 
haps in  function)  the  corpus  luteum  of  the  ovary.  In  some  marine 
animals,  indeed  (eels,  sharks,  etc.),  the  sympathetic  tissue  portion  and 
the  cortical  tissue  exist  as  separate  organs. 

The  adrenal  cortex  seems  to  be  related  especially  to  the  generative 
system,^^  as  shown  by  the  following  facts: 

1.  The  embryologic  origin  in  the  urogenital  anlage,  and  the  histologic  struc- 
ture which  is  similar  to  the  corpus  luteum. 

2.  In  many  animals  there  occurs  hypertrophy  of  the  cortex  during  the  breeding 
season,  and  there  are  histological  differences  in  the  glands  of  males  and  females 
(Kolmer). 

3.  Many  cases  of  sexual  precocity  have  been  observed  in  association  with 
tumors  or  hypertrophy  of  the  adrenal  cortex;  and  defective  sexual  development 
has  been  found  associated  with  atrophy  of  this  tissue.^* 

4.  The  medulla  increases  relatively  little  in  size  after  birth,  while  the  cortex 
increases  with  the  development  of  the  individual. 

Whether  the  cortex  has  other  functions  or  not  is  not  j'et  known. ■'^ 
Biedl  has  found  evidence  that  cortical  substance  is  essential  for  life." 
Animals  with  accessory  adrenals,  which  contain  onlj'  cortical  substance, 
withstand  ablation  of  the  adrenals  proper,  presumably  because  the 

*°  MacCallum,  Johns  Hopkins  Hosp.  Bull.,  1905  (16),  287. 

"  The  calcium  excretion  in  exophthalmic  goiter  parallels  the  nitrogen  (Towles, 
Amer.  Jour.  Med.  Sci.,  1910  (140),  100). 

"  Literature  given  by  Bayer,  Ergebnisse  Pathol.,  1910  (XlVo),  1. 

*^  The  chrome  reaction  (observed  in  the  adrenal  medulla,  carotid  and  coccygeal 
glands  and  the  sympathetic  ganglia),  as  well  as  other  reactions  for  these  tissues, 
is  based  on  reduction  of  chromic  acid  to  chromium  dioxide  by  epinephrine  (Ogata, 
Jour.  Exp.  Med.,  1917  (25),  807). 

^^See  Kolmer,  Pfltiger's  Arch.,  1912  (144),  361;  Vincent,  Surg.,  Gvn.,  Obst., 
1917  (25),  299. 

"  See  Glynn,  Quart.  Jour.  Med.,  1912  (5),  157;  Jump  et  al,  Amer.  Jour.  Med. 
Sci.,  1914  (147),  568. 

••^  It  does  not  have  a  marked  effect  on  the  development  of  tadpoles,  hence  dif- 
fering from  thyroid  and  thymus  (Gudernatsch). 

*' See  also  Crowe  and  Wislocki,  Bull.  Johns  Hopkins  Hosp.,  1914  (25),  287; 
and  Wheeler  and  Vincent,  Trans.  Roy.  Soc.  Canada,  1917  (11),  125. 


616         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

rest  of  the  chromaffin  substance  remains  to  compensate.^*  Chemically 
the  cortex  is  characterized  by  not  containing  the  specific  vaso-constrictor 
principle,  the  epinephrine,  and  by  containing  a  very  large  proportion  of 
lipoids.  Thus,  in  water-free  human  adrenals  (cortex  and  medulla 
both  included)  there  was  found  36.3  per  cent,  of  ether-soluble  material, 
of  which  20.6  per  cent,  was  cholesterol  and  33  per  cent,  was  phospholi- 
pins.*^  The  proportion  of  fats  and  lipoids  varies  greatly  during  changes 
of  age,  disease,  and  perhaps  of  function,  and  there  are  those  who  beheve 
the  adrenal  cortex  to  be  a  chief  source  of  the  hpoids  of  the  blood,  to 
which  much  important  function  is  ascribed  in  the  reactions  of  immu- 
nity. (See  Lipoids,  under  Fatty  Metamorphosis.)  When  cholesterol 
is  fed  in  large  amounts  some  is  deposited  in  the  adrenal  cortex,^" 
while  in  many  diseases,  notably  delirium  tremens  (Hirsch,)^'  the  lipoid 
content  of  the  adrenals  is  greatly  decreased.  In  renal  and  arterial 
disease  the  adrenal  lipoids  have  been  found  increased.^-  The  lipins 
of  the  adrenal  cortex  are  said  to  contain  little  or  no  neutral  fat,*^  but 
free  fatty  acids  which  may  be  increased  when  the  cholesterol  de- 
creases. Loss  of  body  fats  is  not  accompanied  by  a  loss  of  adrenal 
lipoids  ordinarily,  although  they  decrease  in  acute  infections,  especially 
pneumonia. ^^  A  vaso-depressor  effect  is  produced  by  extracts  of  adre- 
nal cortex,  perhaps  caused  by  choline  which  has  been  found  in  such 
extracts,  or  possibly  by  histamine. 

The  medulla  is  characterized,  besides,  by  its  pigmentary  content, 
by  the  remarkably  active  internal  secretion,  epinephrine,^^  which  it 
always  contains  in  greater  or  less  amount.  Presumably  epinephrine, 
of  which  the  formula  is 

ho/     y   HOH— CH2(NH)— CH3 
H0~ 

is  derived  from  the  aromatic  radical  of  the  proteins,  its  close  relation- 
ship to  tyrosine  being  seen  when  the  formula  of  the  latter  is  compared 

H0<^     NcHa— CH(NH2)— COOH 

That  epinephrine  is  formed  from  tjTOsine  directly,  is,  however, 
not  yet  demonstrated.     There  are  also  other  amines  and  aromatic 

^8  See  Fulk  and  MacLoed  (Amer.  Jour.  Physiol.,  1916  (40),  21)  who  found 
that  the  active  principle  of  other  chromaffin  tissues  has  the  same  physiological 
effect  as  that  of  the  adrenal  medulla. 

*'>  Wells,  Jour.  Med.  Res.  1908  (17),  461. 

"'"  Krylov,  Beitr.  path.  Anat.,  1914  (58),  434. 

^'  Jour.  Amer.  Med.  Assoc,  1914  (63),  2186. 

"  Chauffard,  Compt.  Rend.  Soc.  Biol.,  1914  (76),  529. 

"  Borberg,  Skand.  Arch.  Physiol.,  1915  (32),  287. 

"  Elliott,  Quart.  Jour.  Med.,  1914  (8),  47;  Laignel-Lavastine,  Compt.  Rend. 
Soc.  Biol.,  1918  (81),  324. 

''  This  name,  given  by  Abel  and  Crawford,  is  to  be  preferred  to  the  others  in 
common  use,  especially  the  most-used  term  "adremilin,"  which  has  been  copy- 
righted by  a  manufacturing  establishment  so  that  this  name  means  specifically 
their  product,  and  not  the  active  principle  of  the  adrenal  from  whatever  source. 


CHEMISTRY  OF  THE  ADRENALS  (il7 

compounds  whicli  niifrht  he  fornicd  in  tlic  body,  that  lia\'(>  a  i)r('.s.sor 
effect,  and  which  perhaps  are  formed,  although  not  yet  identified." 
It  is  to  be  borne  in  mind  that  the  formation  of  epinephrine  is  not  hmited 
to  the  ach'enals,  but  that  other  ishmds  of  cliromaffin  sympathetic  tissue 
can  do  the  same,"  which  exphiins  tlie  oljserved  discrepancies  Ijetween 
the  anatomic  changes  in  the  adrenals  and  the  clinical  manifestations 
of  a  deficiency  in  epinephrine. 

According  to  Goldzieher^'*  the  normal  human  adrenals  contain  to- 
gether about  4  mg.  epinephrine,  which  may  be  increased  in  conditions 
with  high  blood  pressure,  such  as  arteriosclerosis  and  nephritis,  in 
whicli  he  found  an  average  of  5.8  mg. ;  and  in  septic  conditions  with 
low  pressure  he  found  it  reduced  to  an  average  of  1.5  mg.^"  Lucksch^" 
gives  a  normal  figure  of  4  mg.  for  each  gland,  also  finding  the  amount 
lowered  in  infectious  diseases  and  increased  in  nephritis.  The  human 
adrenal  contains  no  epinephrine  before  birth, ""'^  but  P'enger'^^  found  it 
present  in  the  adrenal  of  unborn  domestic  animals.  Autolysis  of  the 
adrenal  decreases  the  amount,®^  but  not  all  of  the  epinephrine  is 
destroyed  even  several  days  after  death,  as  shown  by  Ingier  and 
Schmorl,*^''  who,  using  both  morphological  and  chemical  methods,  also 
found  a  gradual  increase  in  the  epinephrine  content  of  normal  glands 
from  birth  to  the  ninth  year,  after  which  it  remains  practically  con- 
stant at  about  4.5  mg.  (males  4.4,  females  4.71  mg.).  They  also  found 
a  slight  increase  in  arteriosclerosis,  more  in  acute  and  chronic  nephritits 
and  a  decrease  in  diabetes  and  narcosis,  there  being  practically  no 
epinephrine  in  the  adrenal  of  Addison's  disease.  In  most  of  the  in- 
fectious diseases  they  found  no  changes,  and  in  amyloid  infiltration 
the  amount  was  about  normal.  The  amount  of  chromaffin  substance 
and  epinephrine  do  not  always  run  parallel,  although  Borberg'"'''  found 
a  close  parallelism;  this  author  also  failed  to  observe  any  marked  de- 
crease of  chromaffin  substance  in  narcosis.  Elliott**  found  a  low 
epinephrine  content  in  acute  infectious  diseases,  and  especially  low  in 
acute  cardiac  failure  associated  with  great  mental  distress;  he  did  not 
find  any  increase  in  the  epinephrine  in  nephritis  or  in  any  other  disease. 

The  function  of  the  epinephrine  is  manifestly  to  modify  the  tone  of 
the  non-striated  muscle  fibers  which  are  under  control  of  the  sympa- 
thetic nervous  system,  acting  upon  some  receptive  substance  present 

5«  See  Barger  and  Dale,  Jour.  Physiol.,  1910  (41),  19. 

"  See  Vincent,  Proc.  Rov.  Soc,  B,  1908  (82),  502. 

*»  Wien.  klin.  Woch.,  1910  (23),  809. 

59  See  also  Reich  and  Beresnegowski,  Beitr.  klin.  Chir.,  1914  (91),  403.  Ohno 
(Verh.  Japan.  Path.  GeselL,  1916  (6),  15)  found  the  normal  content  to  be  about 
5.6  mg.  averaging  8.32  mg.  in  chronic  nephritis. 

««  Virch.  Arch.,  1917  (223),  290. 

"  Moore  and  Purinton,  Amer.  Jour.  Physiol.,  1900  (4),  51;  Julian  Lewis,  Jour. 
Biol.  Chem.,  1916  (24),  249. 

62  Jour.  Biol.  Chem.,  1912  (11),  489. 

63  Commessatti,  Arch.  exp.  Path.  u.  Pharm.,  1910  (62),  190. 
«"  Deut.  Arch.  klin.  Med.,  1911  (104),  125. 

«  Skand.  Arch.  Physiol.,  1912  (27),  341;  1913  (28),  91. 


618         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

in  the  muscle,  perhaps  at  the  nerve  endings.  But  it  is  a  fact  of  much 
practical  importance  that  administration  of  epinephrine  will  not 
compensate  successfully  for  the  loss  of  the  adrenals,  whether  because 
the  gland  secretes  other  things,  or  because  the  intermittent  artificial 
administration  of  the  epinephrine  will  not  compensate  for  the  regulated 
secretion  of  the  gland  under  normal  conditions,  or  both.  It  would 
seem  that  the  adrenal  has  an  effect  on  other  glands,  for  injections  of 
epinephrine  cause  glycosuria  in  animals,  as  also  does  manipulation  of 
the  adrenals,  or  painting  the  epinephrine  on  the  pancreas.  There  is 
much  disagreement  as  to  the  effects  of  extirpation  of  the  adrenals  on 
carbohydrate  metabolism,  and  the  nature  and  cause  of  the  effects 
observed.  Biedl  sums  up  the  evidence  with  the  statement  that  the  in- 
ternal secretion  of  the  chromaffin  system  is  of  importance  in  the  mobi- 
lization of  the  sugar  of  the  blood,  and  the  formation  of  the  glj^cogen 
in  the  tissues.  That  the  adrenal  is  at  all  implicated  in  human  diabetes 
has  not  been  demonstrated.  There  seems  to  be  a  relationship  of  mu- 
tual stimulation  between  thyroid  and  adrenal,  for  thyroid  secretion 
sensitizes  the  sympathetic  nerve  endings  to  epinephrine,  and  both 
liberate  carbohydrates  from  the  sugar  depots. ^^ 

Acute  insufficiency  of  the  adrenals,  caused  most  often  by  hemor- 
rhagic infarction,  but  sometimes  by  other  lesions,  may  cause  sudden 
collapse,  asthenia  or  death."  The  extent  to  which  the  cortex  and 
medulla  respectively  are  responsible  is  undetermined.  The  French 
authors  especially  lay  great  weight  on  adrenal  insufficiency  as  a  cause 
of  pathological  states. ^^  Surgical  shock  has  also  been  attributed,  at 
least  in  some  cases,  to  exhaustion  of  the  adrenals,  which  takes  place 
under  the  influence  of  the  anesthetic  and  the  stimulation  to  the  nervous 
system  by  the  operative  manipulation,  perhaps  augmented  by  concur- 
rent infections.''^ 

It  is  possible  that  in  some  cases  of  trauma  to  the  adrenal,  acute 
hemorrhage  or  infection,  intoxication  from  an  excess  of  epinephrine 
might  occur,  but  it  is  improbable  that  fatal  results  could  be  produced 
in  this  way,  for  the  lethal  dose  for  dogs  and  rabbits  is  about  0.1  to  0.25 
mg.  per  kilo,  and  the  two  adrenals  in  man  contain  in  all  but  about 
4  to  5  mg.  epinephrine.  Moderna,  however,  states  that  there  is  so 
much  epinephrine  set  free  after  hemorrhage  into  the  adrenal,  that  it 
can  be  demonstrated  microchemically  in  the  liver,  and  that  the  symp- 
toms and  autopsy  findings  are  identical  with  those  of  acute  epinephrine 
intoxication.  In  animals,  repeated  doses  of  epinephrine  produce 
decreasing  effects,  not  only  on  blood  pressure  but  on  the  glycosuria  and 
other  symptoms,  indicating  an  acquirement  of  tolerance,  but,  because 

«"  See  Endocrinology,  1917  (1),  40-4. 

"Literature  by  Lavenson,  Arch.  Int.  Med.,  190S  (2),  62;  Materna,  Ziegler's 
Beitr.,  1910  (48),  236. 

"8  See  Sergcnt,  Presse  M6d.,  1909  (17),  489;  Cowie  and  Beaven,  Arch.  Int. 
Med.,  1919  (24),  78. 

""See  Ilornowski.  Arch.  m6d.  exp6r.,  1909  (21),  702;  Virchow's  Archiv.,  1909 
(198),  93. 


THE  ADRENALS  AND  ADDISON'S  DISEASE  019 

of  its  nonprotein  nature,  epinephrine  does  not  cause  the  j)ro(luction  of 
antibodies.''" 

Many  studies  have  been  directed  to  determine  the  relation  of  the 
adrenal  to  hypertroi)hy  of  the  heart  and  to  interstitial  nephritis  with 
high  blood  pressure.  Some  have  found  more  or  less  increase  in  size  in 
the  adrenals  under  these  conditions,  chiefly  involving  the  cortex, 
and  a  slight  increase  in  the  epinephrine  content  has  been  reported, 
but  it  is  very  doubtful  if  these  observations  are  of  significance. '''  It 
has  been  reported  by  several  investigators  that  the  blood  in  sucli  condi- 
tions contains  sufficient  epinephrine  to  permit  of  its  detection  and 
measurement  by  its  physiological  effects  (dilatation  of  the  frog's  iris, 
contraction  of  the  rabbit  uterus  or  blood  vessels,  inhibition  of  con- 
traction of  the  intestine).  The  critique  of  this  work  by  Stewart, ^- 
however,  makes  it  necessary  to  discount  most  of  the  published  results, 
as  being  inadequately  controlled.  He  found  no  epinephrine  even  in 
blood  coming  direct  from  the  adrenal  veins,  unless  the  gland  had  been 
stimulated  or  manipulated,  and  none  could  be  detected  in  the  serum 
from  several  patients  with  high  pressure  from  various  causes,  as  well 
as  in  mental  disturbances  and  exophthalmic  goiter.  Vaso-constrictor 
substances  may  be  present  in  serum,  both  normal  and  pathological, 
wliich  are  not  epinephrine.  His  negative  results  are  corroborated  by 
Janeway  and  Park.'^^  Broking  and  Trendelenburg, '"'using  a  perfusion 
method  which  they  believe  to  be  reliable,  found  a  normal  pressor  effect 
from  the  blood  of  persons  with  arteriosclerosis  and  high  blood  pressure, 
a  decrease  in  nephritis  with  high  pressure,  a  great  increase  in  exophthal- 
mic goiter,  and  no  changes  in  pregnancy,  chlorosis  and  diabetes. 

Arterial  Degeneration  from  Epinephrine." — An  interesting  result  of  repeated 
injections  of  epineprhine  into  animals  is  the  appearance  of  a  marked  atheromatous 
degeneration  of  the  aorta,  with  calcification.  This  was  first  observed  by  Josu6, 
and  since  confirmed  by  Erb,  Fischer,  Gouget,  Loeb  and  Githens,  and  many  others. 
These  lesions  are  quite  different  from  those  of  human  aortic  arteriosclerosis,  the 
chief  change  being  degeneration  of  the  muscle-cells  of  the  media,  without  any  con- 
siderable inflammatory  reaction.  There  is,  however,  more  resemblance  to  the 
atheromatous  changes  seen  in  the  arteries  of  the  extremities.  They  do  not  seem 
to  be  due  to  the  heightened  blood  pressure,  since  simultaneous  administration  of 
substances  that  keep  the  blood  pressure  down  does  not  prevent  the  atheroma  from 
developing  (Braun),  while  other  substances  that  raise  blood  pressure,  such  as 
nicotine  (Josuc)  or  pyrocatechin  (Loeb  and  Githens),  do  not  cause  atheroma.  Pre- 
sumably, therefore,  epinephrine  causes  the  arterial  changes  by  a  direct  toxic 
action,  but  the  influence  of  increased  blood  pressure  cannot  be  entirely  excluded. 
However,  slow  injection  of  epinei)hrine,  so  regulated  that  there  is  an  increase  in 
the  blood  content  without  significant  rise  of  pressure,  fails  to  produce  arterio- 
sclerosis.''^    Myocardial  degeneration  is  also  observed  in  experunental  animals, 

7"  See  Elliott  and  Durham,  Jour,  of  Physiol.,  1906  (34),  430. 

"'  See  Pearce,  Jour.  Exper.  Med.,  190S  (10),  735;  Thomas,  Ziegler's  Beitr., 
1910  (49),  228. 

'■'  Jour.  Exp.  Med.,  1911  (14),  377;  1912  (15),  547;  also  Rogoff,  Jour.  Lab. 
Clin.  Med.,  1918  (3),  209. 

"  Jour.  Exp.  Med.,  1912  (16),  541. 

'^Deut.  Arcli.  klin.  Med.,  1911  (103),  168. 

"  Literature  given  by  Saltykow,  Cent.  f.  Path.,  1908  (19),  369. 

'^  van  Leersum  and  Rassers,  Zeit.  exp.  path.,  1914  (16),  230. 


620         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

and  later  may  lead  to  an  interstitial  myocarditis  (Pearce).  These  experiments 
suggest  the  possibility  that  oversecretion  of  epinephrine  may  be  a  cause  of  arterio- 
sclerosis, but  there  is  no  evidence  that  this  actually  occurs  in  man. 

ADDISON'S  Disease" 

As  pointed  out  before,  the  profound  deficiency  in  the  pressor  prin- 
ciples evident  in  the  manifestations  of  Addison's  disease  imphes  loss 
of  function,  not  only  of  the  adrenal  medulla,  but  also  of  the  rest  of 
the  chromaffin  tissues  which  produce  this  same  sort  of  material. 
Therefore  it  is  possible  to  have  any  amount  of  destruction  of  the  ad- 
renals without  Addison's  disease,  if  there  is  sufficient  compensation 
by  the  other  chromaffin  structures,  or,  conversely,  Addison's  disease 
may  occur  when  the  adrenals  seem  morphologically  little  altered,  which 
occurs  in  about  10  per  cent,  of  all  cases.  In  typical  cases,  however 
the  adrenals  have  been  found  entirely  devoid  of  epinephrine,"^  and 
usually  the.  structural  alterations  are  conspicuous.  While  some  have 
held  that  the  destruction  of  the  adrenal  cortex  is  of  importance  in 
Addison's  disease,  this  does  not  seem  to  have  been  conclusively 
demonstrated. 

The  pigmentation  of  the  skin'^^  has  not  yet  been  explained,  but  in 
view  of  the  fact  that  oxidizing  enzymes  readily  convert  epinephrine, 
tyrosine,  and  related  aromatic  substances  into  pigments,  and  that  in 
Addison's  disease  we  have  a  deficiency  in  a  tissue  which  is  known  to  be 
concerned  in  the  metabolism  of  aromatic  compounds,  it  seems  probable 
that  the  pigmentation  is  the  result  of  this  defective  metabolism  of  the 
chromogenic  aromatic  compounds.  In  support  of  this  view  is  the  ob- 
servation of  Bittorf^"  that  the  skin  of  persons  with  Addison's  disease 
has  an  augmented  power  of  oxidizing  epinephrine  and  tyrosine  to  pig- 
mented substances.  Bloch^^  believes  the  pigmentation  to  result  from 
the  presence  of  excessive  quantities  of  3.4-dioxyphenylalanine,  which 
may  be  a  precursor  of  epinephrine,  and  which  is  oxidized  to  a  melanin 

H 
O 

ho/    NcHs.CHNHj— COOH 

by  special  oxidizing  enzymes  ("dopaoxidase")  present  in  the  skin. 
Until  the  pigment  of  Addison's  disease  has  been  isolated  and  analj^zed, 
however,  these  hypotheses  will  probably  remain  unproved.  (See 
pigmentation.  Chap,  xviii.)  Addison's  disease  can  occur  without 
pigmentation. 

That  there  is  a  deficiency  in  the  formation  of  epinephrine  is  at- 

"  Literature  on  Chemistry,  by  EiseU,  Zeit.  klin.  Med.,  1910  (69),  393. 
"  Ingier  and  Schmorl,  Dent.  Arch.  klin.  Mod.,  1911  (101),  125. 
"  According  to  Straub  (Deut.  Arch.  klin.  Mod.,  1909  (97),  07)    pigmentation 
may  occur  within  17  days  after  throndiosis  of  the  adrenal  vein. 
«»  Arch.  exp.  Path.,  1914  (75),  143. 
8'  Zeit.  exp.  Mod.,  1917  (5),  179;  Arch.  f.  Dermatol.,  1917  (124),  h.  2. 


77//';  ini'OI'/lYSI.S  AM)  ACROMEGALY  021 

tested  by  the  low  blood  pressure  and  Kt^ni^i'^il  l^^v  tone  of  the  unstri- 
ated  muscle  tissue.  Carbohydrate  metabolism  is  also  altered,  Porges"'^ 
having  found  hypogluccmia  in  Addison's  disease;,  and  an  increased 
sugar  tolerance  having  been  observed  by  others.  Whether  the  ad- 
renals exert  a  deto.xicating  effect,  and  the  symptoms  of  the  disease 
are  partly  the  result  of  an  autointoxication  of  some  sort,  is  at  present 
unknown,  although  this  idea  has  often  been  advanced.  The  general 
metabolism  of  Addison's  disease  shows  no  very  striking  or  character- 
istic changes,  over  and  above  those  associated  with  the  emaciation. 
Wolf  and  Thacher^'*  found  a  decrease  in  endogenous  creatine  and 
purine  excretion,  and  some  evidences  of  acidosis  towards  the  end  of 
the  disease;  deaminizing  power  and  oxidation  of  cystine  sulphur  to 
SO4  were  not  impaired.  Eiselt  believes  that  there  is  a  toxicogenic 
loss  of  tissue. 

Administration  of  adrenal  tissue  and  extracts,  or  epinephrine, 
whether  by  mouth  or  subcutaneously,  is  not  effective  m  ameliorating 
the  course  of  Addison's  disease,  at  least  in  most  cases.  Thus,  in  97 
cases  collected  bj''  Adams, ^^  adrenal  treatment  caused  some  improve- 
ment in  31,  43  were  not  benefited,  7  were  made  worse,  while  16 
were  described  as  permanently  improved.  The  most  favorably 
affected  is  usually  the  muscular  and  gastro-intestinal  asthenia,  while 
the  pigmentation  is  not  usually  altered.  There  is  little  effect  on 
metabolism.  ^^ 

The  Hypophysis  and  Acromegaly's 

Although  the  hj^pophysis  contains  in  its  anterior  lobe  and  in  the 
pars  intermedia,  a  certain  number  of  spaces  filled  with  colloid  and  re- 
sembling the  alveoli  of  the  thyroid  in  appearance, ^^  there  is  no  evidence 
that  an  appreciable  amount  of  iodin  is  present  here  except  when  thera- 
peutically administered.^^  The  posterior  lobe  contains  an  active  diu- 
retic and  pressor  substance, ^^  the  exact  nature  of  which  is  not  yet 
known,  although  in  many  respects  its  action  resembles  that  of  epi- 
nephrine. It  seems  less  active  in  producing  arteriosclerosis  than  is  epi- 
nephrine, and  its  pressor  effects  are  of  longer  duration.  It  seems  to 
stimulate  smooth  muscle  without  respect  to  innervation  (thus  differing 

82  Zeit.  klin.  Med.,  1909  (69),  341;  also  Bernstein,  Berl.  klin.  Woch.,  1911  (48), 
1794.  Normal  blood  sugar  was  found  by  Broekmeyer,  Deut.  med.  Woch.,  1914 
(40),  1562. 

83  Arch.  Int.  Med.,  1909  (3),  438. 
8*  Practitioner,  1903  (71),  472. 

85  Beutenmliller  and  Stoltzenbergcr,  Biochem.  Zeit.,  1910  (28),  138. 

8«  Full  bibliography  in  the  monograph  by  Harvey  Gushing,  "The  Pituitary 
Body  and  its  Disorders,"  Philadelphia,  1912;  also  .\schner,  Pfluger's  Arch.,  1912 
(146),  1.  ^  o 

87  Composition  of  hypophysis  given  bv  MacArthur,  Jour.  Amer.  Chem.  Soc, 
1919  (41),  1225. 

88  Wells,  Jour.  Biol.  Chem.,  1910  (7),  259. 

89  Lewis,  Miller  and  Matthews,  Arch.  Int.  Med.,  1911  (7),  785;  Herring,  Quart. 
Jour.  Exp.  Physiol.,  1914  (8),  245  and  267. 


622         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

from  epinephrine),  but  with  a  special  potency  in  stimulating  con- 
tractions of  the  uterus;  and  hence  it  has  a  wide  clinical  use  under 
the  name  pituitrin.  The  chemical  nature  of  pituitrin  is  not  yet  deter- 
mined, but  it  seems  to  be  closely  related  to  (8-iminazolylethyla- 
mine,  the  base  derived  from  histidine,^°  and  which  also  stimulates 
uterine  contractions,  but  which  differs  in  causing  bronchial  spasm 
and  urticarial  reactions.  Abel  believes  that  histamine  is  the  chief 
constituent  of  pituitrin,  presumably  associated  with  a  pressor  base  of 
some  sort.  Hanke  and  Koessler,^°"  however,  seem  to  have  demon- 
strated the  absence  of  significant  quantities  of  histamine  in  fresh 
hypophysis.  Injection  of  the  posterior  lobe  extract  lowers  the  assimila- 
tion Hmit  for  carbohydrates  and  causes  glycogenolysis  (Gushing), ^^ 
and  is  a  powerful  galactagogue  (Ott). 

Removal  of  the  anterior  lobe  of  the  gland  in  young  animals  is  fol- 
lowed by  marked  metabolic  and  developmental  changes,  notable  being 
adiposity,  nutritional  changes  in  the  skin  and  its  appendages,  sexual 
inactivity  and  underdevelopment,  subnormal  body  temperature  and 
increased  carbohydrate  tolerance. ^^  These  manifestations  correspond 
to  those  observed  in  certain  human  conditions  (Froehlich's  syndrome)  ^^ 
associated  with  defects  in  the  hypophysis.  Removal  of  the  posterior 
lobe  does  not  produce  any  characteristic  and  constant  effects,  although 
marked  polyuria  and  erotism  have  resulted.  The  anterior  lobe  fed  to 
young  rats  has  a  stimulating  effect  on  growth,  and  especially  on  sexual 
development  and  activity,  while  posterior  lobe  feeding  has  a  retarding 
influence  (Goetsch^*).  Robertson  describes  a  modification  of  growth 
in  mice  fed  anterior  lobe  substance,  which  he  attributes  to  a  specific 
substance,  tethelin,  containing  phosphorus  and  probably  an  iminazolyl 
group,  and  hence  related  to  the  active  constituent  of  the  posterior  lobe, 
although  it  has  no  pressor  effect.^^ 

Puncture  of  the  hypophysis  produces  the  same  effect  as  puncture 
of  Bernard's  diabetic  center  in  the  fourth  ventricle, ^^  and  stimulation 
of  the  gland  has  a  similar  effect,  presumably  because  of  the  secretion 
of  a  glycogenolytic  agent.  A  diminution  of  posterior  lobe  secretion 
occurring  in  certain  conditions  of  hypopituitarism  leads  to  an  acquired 
high  tolerance  for  sugars,  with  the  resultant  accumulation  of  fat.  In 
hibernating  animals,  also,  the  adiposity  and  lowered  temperature  are 
associated  with  hypoplasia  of  the  anterior  lobe  of  the  hypophysis. 
There  also  seems  to  be  some  relation  between  the  hypophysis  and 

90  See  Abel  and  Kubota,  Jour.  Pharm.  Exp.  Ther.,  1919  (13),  243. 

"""Jour.  Biol.  Chem.,  1920. 

"  See  also  Bull.  Johns  Hopkins'  Hosp.,  1913  (24),  40. 

*^  Concerning  metabolism  after  hypophysectomy  see  Benedict  and  Ilomans, 
Jour.  Med.  Res.,  1912  (25),  409. 

^^  Metabolism  in  Frcelich's  syndrome  has  been  studied  by  Rosenbloom  (Intcr- 
stateMcd.  Jour.,  1917  (24),  475)  who  found  a  slight  loss  of  sulphur  and  phosphorus. 

"^  Johns  Hopkins  Hospital  Bulletin,  191G  (27),  29;  Growth  of  tadpoles  is  also 
stimulated  (P.  E.  Smith,  Univ.  Calif.  Publ.  (Physiol.),  1918  (5),  11. 

^•^  Jour.  Biol.  Chem.,  191G  (24),  409. 

»»  Amer.  Jour.  Physiol.,  1913  (31),  xiii. 


THE  HYPOrilYSrS  AND  ACROMEGALY  ()23 

urinary  secretion,  for  extracts  of  the  posterior  lobe  cause  marked  p(;ly- 
uria,  and  in  some  instances  of  "diabetes  insipidus,"  lesions  have  been 
found  in  the  hypophysis.  Simmonds'-*^  holds  that  the  pars  intermedia 
is  responsible.  Like  the  thyroid,  the  hypophysis  enlarges  during 
pregnancy. ^^  Feeding  of  hypophysis  is  said  to  increase  both  gaseous 
and  nitrogenous  metabolism,  and  in  a  case  of  hypopituitarism  the 
urine  has  been  found  to  contain  a  high  proportion  of  undetermined 
nitrogen  and  of  neutral  sulphur.^"  Varying  results  have  been  ob- 
tained in  studies  on  the  basal  metabolism  of  h\'poi)ituitarism.' 

Acromegaly. — The  accumulating  evidence  seems  to  have  practi- 
cally proved  that  acromegaly  depends  upon  a  hyperfunctionating  of 
the  anterior  lobe  tissue  of  the  hypophysis,  one  of  the  most  important 
facts  being  the  improvement  which  has  followed  removal  of  the  hyper- 
plastic tissues  in  several  cases  successfully  operated.  Although  there 
are  many  cases  of  tumor  of  the  hypophysis  without  acromegaly,  this 
is  of  no  significance  since  it  is  not  to  be  expected  that  all  tumors  will 
carry  on  the  functions  of  the  tissue  in  which  they  arise.  Acromegaly 
without  hypophyseal  changes  is  rare,  especially  if  we  consider  the 
finer  cytological  evidence  of  cellular  activity.^  So  far,  little  of  chem- 
ical interest  has  been  learned  concerning  this  disease.  The  metabo- 
lism studies  generally  indicate  a  retention  of  nitrogen,  phosphorus  and 
calcium,  because  of  the  overgrowth  of  bone  and  soft  tissues.'  Ac- 
cording to  some  observers  this  retention  is  decreased,  or  changed  to  an 
excess  elimination,  by  administration  of  hypophyseal  substance. ■•  The 
elimination  of  endogenous  uric  acid  is  said  to  be  greatly  increased 
in  acromegaly,  and  decreased  in  cases  with  hypofunction  of  the 
gland. ^     A  considerable  excretion  of  creatine  was  observed  by  Ellis. ^ 

Glycosuria  and  actual  diabetes  is  frequently  present  in  acromegaly 
(40  per  cent,  of  the  cases  collected  by  Borchardt),^  presumably  from 
interference  with  the  regulating  function  of  the  hypophysis,  but  this 
assumption  has  been  questioned  because  of  the  fact  that  lesions  in 
this  location  might  also  produce  glycosuria  by  affecting  the  "diabetic 
center."  However,  since  puncture  of  the  hypophysis  causes  glyco- 
suria, while  injection  of  posterior  lobe  extract  produces  glj'cosuria 
dependent  upon  hyperglycemia  (Gushing),  and  in  view  of  the  fact 
brought  out  by  Borchardt  that  in  cases  of  tumor  of  the  hypophysis 
without  acromegaly,   glj'cosuria  has  never  been  observed,   there  is 

«"  Munch,  nied.  "Woch.,  1913  (60),  127. 

98  SeeErdheim  and  Stumme,  Ziegler's  Beitr.,  1909  (46),  1. 

99  Stetten  and  Rosenbloom,  Proc.  Soc.  exp.  Biol,  and  Med.,  1913  (10),  100. 

1  Means,  Jour.  Med.  Res.,  1915  (32),  121. 

2  See  Lewis,  Bull.  Johns  Hopkins'  Hosp.,  1905  (16),  157. 

3  See  Bergeim,  Stewart  and  Hawk.  Jour.  Exp.  Med.,  1914  (20),  218. 

^  See  Rubinraut,  Dissert.,  Zurich.  Gebr.  Leeman,  1912;  Medigreceanu  and 
Kristeller,  Jour.  Biol.  Chem.,  1911  (9),  109. 

6  Falta  and  Nowaczvnski,  Bed.  klin.  Woch.,  1912  (49),  1781. 
6  Jour.  Amer.  Med.  Assoc,  1911  (56),  1870. 
'  Zeit.  klin.  Med.,  1908  (66),  332. 


624         CHEMICAL  PATHOLOGY  OF  THE  DUCTLESS  GLANDS 

much  probability  that  in  many  if  not  all  of  the  cases  of  glycosuria  with 
acromegaly,  it  is  the  hypophysis  itself  that  is  concerned,  and  that  both 
the  acromegaly  and  the  glycosuria  are  caused  by  hyperactivity  of  the 
gland. 

In  later  stages  of  acromegaly  there  may  develop  a  hypoactivity 
because  of  pressure  upon  the  posterior  lobe  or  infundibular  stalk, 
whereupon  the  sugar  disappears  and  is  replaced  by  an  increased  toler- 
ance for  sugar,^ 

Thymus^  and  Other  Ductless  Glands 

From  the  chemical  standpoint  little  of  interest  is  known  concerning  this  organ. 
It  is  frequently  used  as  a  source  of  nucleic  acids,  in  which  it  is  rich,  but  there  is  no 
study  of  its  chemical  changes  that  is  of  interest  in  pathology.  Numerous  reports 
have  indicated  that  removal  of  the  thymus  causes  marked  changes  in  ossification  and 
development,  but  the  more  recent  studies  do  not  indicate  that  the  thymus  is 
essential  for  life  or  growth  (Park  and  McClure').  Gudernatsch'"  found  that 
feeding  thymus  to  tadpoles  causes  a  great  increase  in  the  rate  of  growth,  and  de- 
creases or  suppresses  the  developmental  changes,  having  exactly  the  opposite 
effect  from  thyroid  feeding,  and  Abderhalden'i  has  found  that  this  property  persists 
after  digestion  of  the  thymus  tissue.  The  failure  of  metamorphosis  on  thymus 
diet  presumably  depends  on  a  lack  of  some  substance  rather  than  on  the  presence 
of  a  specific  agent  inhibiting  metamorphosis.^^  As  yet  no  substance  has  been  iso- 
lated which  can  be  considered  as  a  specific  internal  secretion  of  the  thymus,  although 
the  frequent  concurrence  of  abnormal  conditions  in  the  thyroid  and  thymus,  in  the 
adrenals  and  thymus,  in  the  hypophysis  and  thymus,  together  with  the  frequency 
of  polyglandular  conditions,  leaves  no  question  that  the  thymus  is  to  be  considered 
with  the  other  members  of  this  system,  however  different  its  histological  structure 
may  be.^^  Thymus  administration  by  mouth  is  said  to  counteract  the  effect  of 
thyroid  feeding  in  stimulating  metabolism. ^^  The  enlargement  of  the  thymus  that 
occurs  in  most  cases  of  exophthalmic  goiter  is  accompanied  at  times  by  symptoms 
that  suggest  an  intoxication  from  this  source.  ^^  Uhlenhuth'^  considers  the  thj^mus 
to  be  antagonistic  to  the  parathyroids  and  responsible  for  tetany  parathyreopriva. 

The  chemistry  of  the  Pineal  Gland  can  be  dismissed  practically  without  con- 
sideration, since  no  positive  facts  have  been  brought  to  light. ^'  Extracts  from  the 
organ  show  no  distinct  physiological  effects.  ^^  Tumors  of  the  pineal  gland  have 
been  found  associated  with  adiposity  and  with  precocious  sexual  development,  but 
whether  from  the  action  of  the  gland  itself  or  from  the  pressure  on  the  brain, 
cannot  be  said.^^     Extirpation  of  the  pineal  seems  to  have  no  noticeable  effects  ot 

8  Full  discussion  in  Johns  Hopkins  Hosp.  Bull,  1911  (22),  165;  1913  (24),  40. 

^  In  addition  to  Biedl's  "Innere  Sekretion,"  see  Wiesel,  Ergebnisse  Physiol., 
1911  (XV  (1)  ),  416;  Park  and  McClure,  Amer.  Jour.  Dis.  Chil.,  1919  (18),  317. 

"Arch.  Entwicklgs.,  1912  (35),  457;  Amer.  Jour.  Anat.,  1914  (15),  431. 

11  Arch.  ges.  Physiol.,  1915  (162),  99. 

1^  Uhlenhuth,  Endocrinology,  1919  (3),  284. 

13  Literature  given  by  Basch,  Zeit.  exp.  Path.,  1913  (12),  180. 

1^  Halverson  ciaZ.,  Arch.  Int.  Med.,  1916  (18),  800. 

"  See  review  by  Halsted,  Bull.  Johns  Hopkins  Hosp.,  1914  (25),  223;  Eddy, 
Canad.  Med.  Assoc.  Jour.  March,  1919. 

i«  Jour.  Gen.  Physiol.,  1918  (1),  23;  Endocrinology,  1919  (3),  285. 

1'  Bibliography  by  McCord,  Trans.  Amer.  Gyn.  8oc.,  1917  (43),  109;  Gord  on, 
Endocrinology,  1919 \3),  437. 

1*  Jordan  and  Eyster,  Amer.  Jour.  Physiol.,  1911  (29),  115;  Dixon  and  Halli- 
burton, (^lart.  Jour.  Exper.  Physiol.,  1909  (2),  283.  Dana  and  Berkeley,  Med. 
Record,  1913  (83),  No.  19. 

19  aee  Pappenheimer,  Virchow's  Arch.,  1910  (200),  122. 


THE  CAROTID  GLAND  025 

any  sort  (Dandy**)  although  McCord"  reports  increased  growth  and  early  sexual 
maturity  in  animals  fed  pineal  substance.** 

The  Carotid  Gland  is  more  directly  related  to  the  adrenal  medulla,  in  that  it 
contains  chromaffin  tissue.  It  should,  presumably,  contain  a  pressor  principle,  as 
Moulon  found,  but  Gomez*^  obtained  only  lowerinp;  of  blood  pressure  from  extracts 
of  this  gland,  and  bilateral  removal  causes  no  characteristic  effects.** 

*»  Jour  Exp.  Med.,  1915  (22),  237. 
*'  Jour.  Amer.  Med.  Assoc,  1914  (63),  232  and  517. 

**  Concerning  the  composition  of  the  pineal  gland  see  Fenger,  Jour.  Amer.  Med. 
Assoc.  1916  (67),  1836. 

*'  Amer.  Jour.  Med.  Sci.,  July,  1908. 

2*  Massaglia.  Frankf.  Zeit.  Path.,  1916  (18).  333. 


40 


CHAPTER  XXIII 

URIC-ACID   METABOLISM  AND   GOUTi 

These  subjects  have  been  the  object  of  such  a  prodigious  amount 
of  research  that  it  is  far  beyond  the  scope  of  this  work  to  review  the 
history  and  the  details  of  the  investigations.  Such  a  review  is  also 
particularly  unnecessary,  since  it  can  be  found  in  the  works  on  phys- 
iological chemistry  and  various  treatises  on  metabohsm.  Conse- 
quently the  attempt  will  be  made  in  this  chapter  merely  to  give,  as 
briefly  as  possible,  the  views  now  most  generally  accepted  concerning 
the  nature  and  metabolism  of  uric  acid,  and  its  relation  to  patho- 
logical processes.  For  the  historical  discussion,  indicating  by  what 
devious  steps  we  have  reached  our  present  understanding  concerning 
this  long-disputed  subject,  the  reader  is  referred  to  the  articles  men- 
tioned below,  upon  which  I  have  drawn  freely.  A  particularly  clear 
summary  of  the  subject  is  given  by  Walter  Jones  in  his  monograph  on 
nucleic  acids. ^ 

THE  CHEMISTRY  OF  URIC  ACID 

It  is  the  very  great  service  of  Emil  Fischer  to  have  shown  us  the 
structure  of  the  uric-acid  molecule,  the  empirical  formula  of  which, 
C5H4N4O3,  had  long  been  known.  He  demonstrated  that  it  is  a  mem- 
ber of  a  group  of  substances,  which  are  all  characterized  bj'  being 
built  up  about  a  certain  nucleus,  C5N4.  As  the  simplest  member  of 
the  group  is  a  synthetically  formed  body,  purine,  the  nucleus  is  called 
the  '^-purine  nucleus."  The  structural  relations  of  the  better-known 
^'purine  bodies"  to  this  purine  nucleus  and  to  each  other  are  clearly 
shown  by  their  structural  formulae,  as  given  below: 

The  atoms  in  the  "purine  nucleus"  are  arranged  as  follows: 
N(i)-C(6) 

C(2)  -  C(5)  -N(7) 
N(3)-C(4)-N(9) 

To  each  atom  has  been  given  a  number,  as  shown,  for  the  purpose  of 
facilitating  reference  to  the  location  of  various  atoms  and  groups 

^  Complete  reviews  are  given  by  F.  H.  McCrudden,  "Uric  Acid,"  New  York, 
1906;  Wiener,  Ergebnisse  der  Physiol.,  1902  (1),  555;  ibid.,  1903  (2),  377;  Burian 
and  Schur,  Pfliiger's  Arch.,  1900  (80),  241;  1901  (87),  239;  Schittenhelni,  Handb.  d. 
Biochem.,  1910,  IV  d),  489;  Brugsch  and  Schittenhelni,  "Die  Nukleinstoffwoclisol 
und  seine  Storungen,"  Jena,  1910;  Walter  Jones,  "Nucleic  Acids,"  Monographs  on 
Biochemistry,  1914.  An  excellent  summary  of  recent  work  is  given  by  Benedict, 
Jour.  Lab.  Clin.  Med.,  1916  (2),  1. 

626 


CHEMISTRY  OF  Till-:  PURINES 


()27 


that  arc  attached  to  this  nucUnis.     The  structure  of  purine  itself  is 
as  shown  below  :- 

N  =  CH 

I  I 

HC    C-NH 

II  II       >" 
N-C-N 

Purine 

The  derivatives  of  purine  are  described  by  stating  to  which  atom 
of  the  purine  nucleus  the  combining  groups  are  attached.  Thus, 
adenine  is  referred  to  as  6-amino-purinc,  and  therefore  has  the  follow- 
ing formula : 

N  =  C-NHo 

I       I 
HC     C-NH 

II   I!  >H 

N-C-N 

Adenine  (G-amino-purine) 

Other  important  members  of  this  group  of  "purine  bodies,"  (also 
called  xanthine  bodies,  alloxuric  bodies,  and  nuclein  bodies)  are  built 
up  about  the  purine  nucleus  as  shown  below: 
HN-C=0 


H2N-C    C-NH 


,11      II       ^ 
N-C-N 

Guanine 
(2-amino-6-oxy  purine) 

HN-C=0 


CH 


HC 


C-NH 
^  CH 


l|       i"      ^ 
N-C-N 

Hypoxanthine 
(C-oxy  purine) 

H3C-N-C=0 

! 

0=C     C-N/ 

I     II      v 


CH3 

CH 


H3C-N-C-N 

Caffeine 
(1-3-7  trimethyI-2-6  dioxy purine) 


HN-C=0 
0=C     C-NH 

J   i:     >CH 

HN-C-N 

Xanthine 
(2,  6-dioxypurine) 

HN-C=0 
0=C     C-NH 

\c=o 

HN-C-NH 

Uric  acid 
(2-0-S-trioxy  purine) 

HN-C  =  0     CH3 

i       1  / 

0=C      C-N/. 

I        II  /^^ 

H,C-N-C-N 

Theobromine 
(3-7-dinicthyI,  2-6  dioxy  purine) 


As  shown  by  their  structural  formulae,  the  pyrimidines  present  in 
the  nucleic  acids  are  also  closely  related  to  the  purines,  viz: 
N-CH  N-C-NH2  HN-C=0  HN-C=0 

II    II  II-  I    ; 

0=C    C-CH, 


HC     CH 


.i> 


N  =  CH 

Pyrimidine 


0  =  C     CH 

I       H 
HN-CH 

Cytosine 

(2-oxy,  G-amino- 

pyrimidine) 


0=C     CH 

I        ' 
HN-CH 

Uracil 

(2-6-dioxy- 
pyrimidine) 


HN-CH 

Thymine 

(5-methyl,  2-6-dioxy- 

pyrimidine) 


2  In  these  formula)  the  symbols  of  the  atoms  forming  the  purine  nucleus  are 
in  heavy  type. 


628  URIC-ACID  METABOLISM  AND  GOUT 

Properties  of  Uric  Acid. — Uric  acid,  when  pure,  is  white,  and  crystallizes  in 
rhombic  tablets.  Its  solubility  is  very  slight;  at  room  temperature  (18°)  it  dissolves 
but  about  one  part  to  40,000  of  water,  so  that  a  saturated  solution  contains  but 
0.0253  gram  to  the  liter.  It  is  much  more  soluble  in  blood-serum,  dissolving  in 
1000  parts,'  probably  held  in  some  complex  combination.  His  and  Paul  have 
shown  that  in  a  saturated  solution  only  9.5  per  cent,  of  the  molecules  are  disso- 
ciated, the  dissociation  occurring  in  two  steps;  the  first  and  chief  dissociation  is 
into  H  and  C.SH3N4O3,  which  then  undergoes  further  dissociation  into  H  and 
C5H2N4O3,  the  latter  dissociation  being  very  slight.  If  any  other  acid  is  present 
in -the  solution,  its  dissociation  and  liberation  of  free  hydrogen  ions  interferes  with 
the  dissociation  of  the  uric  acid,  and  as  the  undissociated  uric  acid  is  extremely 
insoluble,  the  amount  dissolved  in  an  acid  solution  is  much  less  than  in  a  neutral 
solution.^ 

Gudzent^  found  that  saturated  solutions  of  urates  gradually  precipitate  out  the 
salts  because  of  a  transformation  of  part  of  the  uric  acid  into  what  he  believes  to 
be  a  lactirn  form.  (The  lactim  form  is  shown  in  the  following  formula,  as  com- 
pared with  the  isomeric  lactam  form  shown  above,  in  which  uric  acid  is  supposed 
to  exist  ordinarily.) 

N=C— OH 
I       I 
HO— C     C— NH 

\ 
C— OH 

/- 

N— C— N 

(Lactim  form  of  uric  acid) 

With  alkalies  uric  acid  yields  two  series  of  salts,  corresponding  to  these  two 
steps  in  dissociation:  one,  in  which  one  atom  of  the  base  enters,  is  called  the 
biurate  or  monobasic  urate;  the  other  is  the  so-called  "neutral"  or  bibasic  urate. ^ 
Of  the  two,  the  latter  is  much  the  more  soluble.  The  monosodiumtirate  forms 
colloidal  solutions  in  water,  from  which  the  crystalline  salt  gradually  falls  out. 
The  quadriurate,  of  which  much  was  said  in  the  earlier  literature,  probably  does 
not  exist  (Kohler).' 

In  the  urine  the  uric  acid  and  the  urates  are  kept  in  solution  by  the  phosphates, 
the  disodium  phosphate  preventing  the  decomposition  of  the  urates  into  uric  acid  by 
the  acid  salts  of  the  urine.  Possibly  other  constituents  of  the  urine,  especially 
the  pigments  and  NaCl,  also  aid  in  its  solution.  Urine  may  form  quite  stable 
supersaturated  solutions  and  Kohler  states  that  the  urine  is  a  truly  supersaturated 
solution  of  sodium  urate.  How  the  uric  acid  is  kept  in  solution  in  the  blood  is  not 
exactly  understood,  but  Gudzent  believes  that  uric  acid  can  exist  in  the  blood  only 
as  the  monosodium  urate,  and  in  the  less  soluble  but  more  stable  lactim  form,  which 
is  soluble  only  to  the  extent  of  8.3  mg.  per  100  c.c.  .serum  (the  lactam  form  being 
soluble  up  to  18  mg.).  However,  amounts  over  20  mg.  per  100  c.c.  have  been 
detected  in  the  blood  of  nephritics;  here  solution  may  have  been  aided  by  the 
other  retained  metabolites.  Bechhold'  and  others  have  maintained  that  urates 
may  be  present  in  the  blood  in  a  colloidal  state  which  cannot  pass  out  through 
the  kidneys. 

FORMATION  OF  URIC  ACID^' 

The  origin  of  uric  acid  is  chiefly,  although  not  exclusively,  from  the 
nucleoproteins,  and  it  is  customary  to  refer  to  uric  acid  formed  from 
the  nucleoproteins  of  the  foods  as  ''exogenous''  uric  acid,  in  contrast 

»  Taylor,  Jour.  Biol.  Chem.,  1906  (1),  177. 

*  Concerning  the  solubility  of  uric  acid  in  urine  see  Haskins,  Jour.  Biol.  Chem., 
1916  (26),  205. 

"  Zeit.  physiol.  Chem.,  1909  (60),  38. 

"  As  a  matter  of  fact,  both  salts  give  a  slightly  alkaline  reaction  when  dis- 
solved in  water  (Taylor). 

'Zeit.  physiol.  Chem.,  1911  (72),  169;  1913  (78),  205;  Zeit.  klin.  Med.,  1919 
(87), f  338. 

»  Biochem.  Zeit.,  1914  (64),  471. 

»  See  review  in  International  Clinics,  1910,  XX  d),  76. 


CHEMISTRY  OF  NUCLEIC  AC  J  I)  V,2\) 

to  the  "endogenous"  uric  acid  tliat  is  lormed  from  the  imclco-|)r()t(!ins 
of  the  body  cells  during  their  catabolism.  This  may  be  readily 
explained  by  a  brief  considciration  of  the  composition  of  the  nudeo- 
proteius.  The  nucleoprotcins  may  be  looked  upon  as  salts  formed 
through  combination  of  proteins  with  nucleic  acid.  Nucleic  acid  in 
turn  is  a  compound  of  phosphoric  acid  with  purine  bases,  pyrimidine 
bases,  and  carbohydrate  radicals,  constituting  a  complex  sort  of  glu- 
coside. 

A  long  series  of  careful  analytical  studies  has  at  last  shown  us  that 
nucleic  acids,  are,  whatever  the  source,  quite  similar  in  composition, 
consisting  always  of  a  complex  containing  phosphoric  acid,  the  two 
amino  purines  (adenine  and  guanine),  two  pyrimidines  (either  cyto- 
sine  and  uracil  or  cytosine  and  thymine);  and  a  carbohj'drate,  which 
may  be  either  a  pentose  or  a  hexose.  Apparently  there  arc  two  sorts 
of  nucleic  acids,  one  from  plants,  wliich  contains  always  uracil  and 
pentose,  and  one  from  animal  tissues,  containing  instead  thymine  and 
a  hexose.  So  constant  are  the  findings  in  regard  to  these  compounds 
that  it  has  seemed  feasible  to  consider  their  manner  of  union  in  the 
intact  nucleic  acid  molecule,  and  Levene  and  Jacobs  have  proposed  as 
the  structure  of  thymus  nucleic  acid  the  following  arrangement : 
H  PO3— C6H10O4— CsHsNsO 

I  (guanine  group) 

o 

H2PO4  -CeHsOo— C5H5N2O2 

I  (thymine  group) 

o 

I 

H2PO4  -CsHsOo— C4H,N30 

(cytosine  group) 

o 

I 

H  PO3 — C6H10O4 — C6H4N6 

(adenine  group) 

It  will  be  seen  that  this  proposed  formula  postulates  in  the  nucleic 
acid  molecule,  one  radical  of  each  of  the  two  purines  and  pyrimidines, 
each  of  these  being  united  by  a  carbohydrate  radical  to  a  phosphoric 
acid  radical.  Recognizing  that  this  must  be  looked  upon  as  a  provi- 
sional formula,^"  it  will  serve  as  a  base  of  departure  from  which  to 
consider  the  metabolism  of  nucleic  acid. 

The  grouping  of  hexose  +  purine  or  hexose  +  pyrimidine  is  re- 
ferred to  as  a  "nucleoside,"  analogous  in  terminology  to  "glucoside." 
The  same  groupings  plus  the  phosphoric  acid  radical  constitute  the 
"nucleotids,"  nucleic  acid  thus  being  made  up  of  four  nucleotids. 
Emil  Fischer  has  reported^^  the  synthetic  production  of  a  nucleotide 
composed  of  phosphoric  acid  united  to  a  glucoside  of  theophyllin,  this 

"  Jones  and  Read  (Jour.  Biol.  Chem.,  1917  (29),  111)  have  advanced  evidence 
to  indicate  that  the  linkage  between  the  nucleotids  is  between  the  carbohydrate 
radicals  rather  than  between  the  phosphoric  acid  groups.  See  also  Thannhauser 
and  Dorfmiiller,  Zeit.  physiol.  Chem.,  1917  (100),  121. 

"  Sitzungsber.  k.  .\kad.  Wissensch.,  Berlin,  1914  (33),  905. 


630  URIC-ACID  METABOLISM  AND  GOUT 

really  constituting  the  long-sought  synthesis  of  a  nucleic  acid,  even 
though  the  artificial  product  is  not  the  same  as  any  known  to  occur  in 
nature.  With  these  facts  before  us  we  may  consider  the  manner  in 
which  nucleic  acids  are  disintegrated  in  the  animal  body. 

So  large  a  molecule  can  conceivably  be  disintegrated  in  many  differ- 
ent ways;  that  is,  the  lines  of  cleavage  might  pass  through  several 
different  points  and  in  many  different  orders,  but  there  is  evidence 
available  which  causes  us  to  beheve  that  the  process  is  quite  constant 
in  animal  metabolism.  Jones  considers  it  probable  that  the  first  step 
is  a  decomposition  of  the  tetranucleotid  into  dinucleotids,  and  that 
these  are  in  turn  spKt  into  mononucleotids.  Little  is  known  about  the 
subsequent  career  of  the  two  pyrimidine  nucleotids,  but  we  have  an 
abundance  of  information  concerning  the  nucleotids  containing  the 
purines,  and  it  is  in  these  our  present  interest  lies.  Each  nucleotid 
has  two  points  at  which  it  might  be  split,  and  we  have  reason  to  believe 
that  there  exist  in  animal  tissues  enzymes  which  may  specifically  at- 
tack each  bond.  One  enzyme  separates  the  phosphoric  acid  radical 
from  the  nucleoside,  thus: 

H2PO4-C6H8O3-C5H4N5O    +  H2O ^H3P04    +  C5H9O4-C6H4N6O 

guanylic  acid  phospho-nuclease  guanosine 

and  this  enzyme  is  therefore  designated  as  phospho-nuclease. 

Another  enzyme,  purine  nuclease,  splits  off,  instead,  the  purine  radi- 
cal, thus: 

H2PO4  -  CsHsOs  -  C5H4N6O  +  H2O >  H.,P04-C6H904  +  CsHsNsO 

guanylic  acid  purine-nuclease  guanine 

Following  either  of  these  cleavages,  the  enzymes  which  deaminize 
purines  begin  to  act,  and  we  have  formed  as  a  result  either  the  free 
oxypurines  or  the  oxypurines  still  bound  in  the  glucoside-like  combi- 
nation with  sugar.     If  the  purines  are  free  the  reaction  will  be : 

C6H5N5O  +  H2O >  C6H4N4O0  +  NH3 

guanine  guanase  xanthine 

or,  in  case  the  guanine  glucoside  is  present: 

C5H9O4  -  C6H4N5O  +  H2O — » C6H9O4  -  C6H3N4O2  +  NH 

guanosine  guanosine-deaminase  xanthosine 

In  the  latter  case  a  hydrolytic  enzyme,  xanthosine-hydrolase,  then 
splits  off  the  xanthine,  so  that  by  either  route  the  end  result  is  the  same. 
By  a  similar  series  of  changes  the  adenine  radical  is  converted  into 
hypoxanthine,  either  directly  by  adenase: 

CsHsNc  +  H2O >  C6H4N4O  +  NH3 

adenine  adenase  hypoxanthine 

or   by  adenosine-deaminase  the  hypoxanthine-glucoside  (inosine)  is 
formed,  and  later  the  hypoxanthine  is  split  off. 


FORMATION  OF  URIC  ACID 


631 


We  now  have  hypoxanthino  and  xantliino,  wliich,  in  the  presence  of 
oxygen,  arc  oxidized  to  form  uric  acid,  thus: 

CsH.N^O  +  O ►  C4H,N«02 

hypoxanthino     bypoxanthinc-oxidoso     xanthine 

CsH.N.O.  +  O ►C5H4N4O, 

xanthine  xanthine-oxidase         uric     acid 

Further  oxidation  of  the  uric  acid  causes  its  conversion  into  the 
much  more  soluble  allantoin,  thus: 


C5H4N4O3  +  O  +  HoO 

uric  acid 


-  CJIeX.Oj  +  CO, 
allantoin 


It  is  thus  evident  that  the  steps  of  the  disintegration  of  nucleic  acid 
are  numerous,  but  that  each  separate  process  is  a  simple  one;  and  also, 
that  it  has  been  possible  to  follow  out  and  distinguish  the  several  steps 
and  to  establish  the  fact  that  each  step  depends  on  a  distinct  and  specific 
enzyme.  Not  every  tissue  possesses  all  the  enzymes  of  purine  destruc- 
tion, and  in  different  species, of  animals  the  distribution  of  the  enzymes 
is  different.  For  example,  the  enzyme  xanthine-oxidase,  which  oxi- 
dizes xanthine  into  uric  acid,  is  found  in  man  only  in  the  liver,  and  also 
in  other  animals  it  is  of  limited  distribution,  being  found  usually  only 
in  the  liver  or  in  the  liver  and  kidney,  but  in  the  dog  it  seems  to  be 
present  in  several  tissues.  The  deaminizing  enzymes,  adenase  and 
guanase,  are  much  more  widely  distributed,  but  by  no  means  univer- 
sally. Adenase,  for  example,  is  not  present  in  the  tissues  of  the  rat, 
and  not  in  the  tissues  of  adult  human  beings. ^^  Guanase  is  absent 
from  the  spleen  and  liver  of  the  pig  and  from  human  spleen,  although 
present  in  most  other  tissues.  Uricase,  the  enzyme  wliich  destroys 
uric  acid,  also  has  peculiarities  of  distribution,  being  seldom  found 
in  any  other  tissue  than  the  liver  or  kidney,  and  being  absent  entirely 
from  the  tissues  of  man,  and  from  the  birds  and  reptiles  so  far  exam- 
ined. The  significance  of  this  distribution  of  uricase  will  be  discussed 
at  greater  length  a  little  later. 

The  following  graphic  expression  of  the  series  of  steps  leading  to 
the  formation  of  uric  acid  has  been  presented  by  Aniberg  and  Jones ;^^ 

OH 
O^P-O.CjHjOj.  CjHN.'OH 

0=P-O.CsH,Oj     CjH,N,(NHj') 
OH 

i^ ^-^-^  i^Phospho-n^lcase      Pho 

5"°"'"*  guonoslne 

C,HN/0*^  .C;H,0, 


odenint 
C,H,N»CHJHi)C,H,0,        C,H,N,(NHO 


/OH 
C,HN-OH 


acid     /  Xontlllnt      y         *: 

-      ^  ox\<ia3*     AanTh 


C.H,N,<0[] 


lypousnrhintf 
C,H,N^OH 


'-  There  have  been  some  reports  indicating  the  presence  of  adenase  in  fetal 
human  tissues  (Long,  Jour.  Biol.  Chem.,  1913  (15),  449). 
"  Zeit.  physiol.  Chem.,  1911  (73),  407. 


632  URIC-ACID  METABOLISM  AND  GOUT 

Another  possible  source  of  uric  acid  is  through  synthesis.  In 
birds,  which  ehminate  most  of  their  nitrogen  in  the  form  of  uric  acid, 
synthesis  of  uric  acid  undoubtedly  occurs.  It  must  also  be  considered 
that  young  mammals  can  synthesize  the  purines  necessary  for  their 
growth  from  foods  which  contain  no  purines. ^^  It  would  seem  possi- 
ble, therefore,  for  synthesis  of  uric  acid  to  occur  in  adult  mammals, 
but  as  yet  satisfactory  experimental  evidence  is  lacking  that  such 
synthesis  does  occur,  although  an  apparently  reversed  reaction, 
whereby  uric  acid  destroyed  by  liver  tissue  can  be  resynthesized  by 
the  same  tissue  acting  upon  it  in  the  absence  of  oxygen,  has  been  de- 
scribed by  Ascoli  and  Izar.^^  Their  work  has  not  been  repeated  suc- 
cessfully by  others.  I  have  failed  in  several  attempts  to  secure  re- 
synthesis  of  uric  acid  by  dog  livers,  and  Spiers,^''  who  made  a  more 
extensive  investigation,  was  unable  to  corroborate  their  findings. 

It  should  also  be  mentioned  that  not  all  of  the  purine  bases  of  the 
body  is  bound  in  the  form  of  nucleic  acid.  A  considerable  amount  is 
present  in  a  free  condition,  or  at  least  not  bound  in  nucleic  acid,  espe- 
cially in  muscle  tissue.  Uric  acid  can  be  formed  as  well  from  the  free 
purine  bases  as  from  purine  bases  liberated  from  nucleic  acid — indeed, 
evidence  has  been  brought  forward  indicating  that  a  large  proportion 
of  the  uric  acid  arising  during  metabolism  (endogenous)  comes  from 
the  free  hypoxanthine  of  the  muscles. 

As  to  the  place  where  uric  acid  is  formed,  it  seems  probable  that  in 
different  animals  different  organs  are  chiefly  concerned,  for  it  has 
been  found  that  the  distribution  of  the  enzymes  mentioned  above 
varies  greatly  in  the  various  orgaps  and  tissues  of  dijfferent  species.  ^^ 
In  most  animals  the  xanthine  oxidase,  which  forms  uric  acid  from  xan- 
thine, is  localized  chiefly  or  solely  in  the  liver,  and  this  is  the  case  in 
man;  therefore  it  is  presumable  that  uric  acid  is  formed  chiefly  in  the 
liver  from  purines  by  the  steps  described  above.  That  there  may  be 
other  methods  of  forming  uric  acid  is  possible. 

DESTRUCTION  OF  URIC  ACIDi* 

With  most  mammals  but  little  of  the  total  amount  of  purine  bases 
taken  as  food  or  set  free  in  the  tissues,  appears  in  the  urine  as  uric 
acid,  most  of  it  being  converted  into  allantoin,  which  seems  to  be  ex- 
creted with  little  or  no  loss.^'-*  Thus,  when  dogs,  pigs  or  rabbits  are 
fed  nucleic  acid,  about  93  to  95  per  cent,  can  be  recovered  as  allantoin, 
3  to  6  per  cent,  as  uric  acid,  and  1  to  2  per  cent,  as  i)urine  bases  (Schit- 
tenhelm).     It  would  seem  that  practically  all  the  purines  can  be  found 

"  McCollum,  Amer.  Jour.  Physiol.,  1909  (25),  120. 

"See  Zeit.  physiol.  Chem.,  1910  (65),  78. 

'SBiochem.  .lour.,  1915  (9).  837. 

1' A  compilation  of  this  distribution  is  given  by  Wells,  Jour.  Biol.  Chein.,  1910 
(7),  171. 

'*  See  discussion  by  Wells,  Jour.  Lab.  Clin.  Med.,  1915  (1),  104. 

"  Allantoin  may  be  found  in  the  blood  of  other  nuimmals  but  not  in  man 
(Hunter,  Jour.  Biol.  Chem.,  1917  (28),  369). 


DESTRUCTION  OF  URIC  ACID  033 

in  these  three  forms  combined,  the  proportions  varying  in  (hfTerent 
species.  In  man  alone,  except  for  the  chimpanzee-'"  and  oraiiK-utan, 
does  a  considerable  proportion  escape  as  uric  acid,  a  fact  in  complete 
harmony  with  repeated  observation  that  the  tissues  of  man  hav(!  no 
power  whatever  to  destroy  uric  acid  in  vitro;  the  earlier  reports  of 
positive  uricolysis  undoubtedly  being  erroneous.  Even  the  monkey 
has  active  uricolytic  enzymes  in  its  hver,  and  therefore  e.xcrotos  its 
purines  chiefly  as  allantoin.  With  mammals  as  a  whole,  therefore, 
uric  acid  is  destroyed  to  the  extent  of  being  converted  into  allantoin,-' 
the  close  relationship  of  which  to  uric  acid  is  shown  by  the  structural 
formula : 

NH  -CH-NH 

0=C  C=0 

NH2     CO  -NH 

(allantoin) 

With  most  mammals  the  oxidation  of  uric  acid  takes  place  chiefly  in 
the  liver,  but  in  some  of  the  herbivora  the  kidneys  are  more  active,  as 
far  as  experiments  in  vitro  can  show. 

Whether  man  can  defetroy  uric  acid  at  all  has  been  a  matter  of  dis- 
pute. It  has  been  shown  by  Wiechowski  and  others  that  uric  acid 
injected  subcutaneously  is  excreted  almost  quantitatively  and  un- 
changed in  the  urine.  To  be  sure,  human  urine  does  contain  a  very 
little  allantoin,  7  to  14  mg.  per  day,  but  this  amount  is  too  small  to  be 
of  much  significance,  for  it  is  possibly  all  derived  from  the  food,  as 
the  human  organism  cannot  destroy  allantoin. 2-  On  the  other  hand, 
it  has  been  found  repeatedly  that  nucleic  acid  or  purines  given  by 
mouth  are  by  no  means  quantitatively  excreted  in  the  urine,  even 
when  uric  acid,  allantoin  and  purine  bases  are  added  together.  Ap- 
parently a  considerable  proportion  of  the  purine  nitrogen  fed,  about 
half  in  most  experiments,  is  excreted  as  urea.-^  As  allantoin  seems 
not  to  be  at  all  disintegrated  in  the  human  body  it  would  seem  prob- 
able that  if  purines  are  destroyed,  as  these  experiments  indicate,  they 
pass  through  some  other  route  than  allantoin,  and  possibly  that  part 
of  the  purines  which  is  destroyed  does  not  pass  through  the  stage  of 
uric  acid.  Experiments  show  that  outside  the  body  uric  acid  can  be 
destroj^ed  by  other  routes  than  through  allantoin;  thus,  it  can  be  dis- 
integrated into  glycine,  ammonia  and  CO2;  or  by  another  method  of 
destruction  it  yields  first  alloxan  (C4H0N2O4),  then  parabanic  acid 
(C3H2N2O3)  which  in  turn  yields  oxalic  acid  and  urea.     There  is  no 

20  Wiechowski,  Prager  med.  Woch.,  1912  (37),  275;  Wells  and  Caldwell,  Jour. 
Biol.  Chem.,  1914  (18),  157. 

21  See  Hunter  and  Givens,  Jour.  Biol.  Chem..  1914  (18),  403. 

22  See  Ackroyd,  Biochem.  Jour.,  1911  (5),  217,  400,  442. 

"  See  Taylor,  Jour.  Biol.  Chem.,  1913  (14),  419;  Givens  and  Hunter,  ibid.,  1915 
(23),  299.  About  one-tenth  as  much  uric  acid  is  excreted  in  the  sweat  as  in  the 
urine,  sweat  containing  0.1  mg.  per  c.c.  (Adler,  Deut.  Arch.  kUn.  Med.,  1916 
(119),  548). 


634  URIC-ACID  METABOLISM  AND  GOUT 

evidence,  however,  that  any  of  these  alternative  routes  is  ever  fol- 
lowed in  the  animal  body.  It  'is  possible  that  the  failure  to  find  all 
the  purines  of  the  food  as  uric  acid  in  the  urine  depends  on  their  par- 
tial destruction  in  the  intestine  by  bacteria.-*  It  is  highly  probable, 
in  view  of  all  available  evidence,  that  in  man  most  of  the  purine  ab- 
sorbed from  the  food,  and  practically  all  the  purine  from  cell  metabo- 
lism, is  converted  into  uric  acid  and  excreted  as  such. 

THE  OCCURRENCE  OF  URIC  ACID  IN  THE  BLOOD,  TISSUES,  AND  URINE 

As  can  be  seen  from  the  foregoing  discussion,  the  amount  of  uric 
acid  that  appears  in  the  urine  depends  upon  a  number  of  factors, 
which  may  be  enumerated  as  follows:-^  (1)  The  amount  of  purine 
bodies  taken  in  the  food,  upon  which,  chiefly,  depends  the  amount  of 
exogenous  uric  acid.  (2)  The  amount  of  destruction  of  tissue  nucleo- 
proteins.  (3)  The  amount  of  purine  bases  formed  in  the  muscle  tis- 
sue. (4)  The  amount  of  conversion  of  purine  bases  into  the  uric 
acid.  (5)  The  amount  of  destruction  of  uric  acid,  if  any,  occurring 
in  the  body.  (6)  Possibly  upon  the  capacity  of  the  tissues  to  synthe- 
size uric  acid;  and  in  case  such  power  to  synthesize  uric  acid  exists, 
upon  the  presence  of  the  precursors  of  uric  acid  in  the  body.  (7)  The 
retention  of  uric  acid  in  the  blood  and  tissues.  (8)  The  power  of  the 
kidneys  to  excrete  uric  acid. 

If  we  also  take  into  account  the  fact  that  the  solubility  of  uric  acid 
in  the  urine  depends  chiefly  upon  the  amount  of  neutral  phosphates 
present  in  the  urine,  and  also  upon  the  temperature,  reaction,  and 
concentration  of  the  urine,  it  becomes  apparent  how  totally  devoid  of 
significance  is  the  presence  of  crystals  of  uric  acid  and  urates  in  the 
urine,  and  how  fallacious  is  any  theorization  based  upon  the  excretion 
of  considerable  quantities  of  uric  acid  when  all  the  above-mentioned 
factors,  especially  the  diet,  are  not  controlled  and  taken  into  consid- 
eration. Yet  on  just  such  an  inadequate  basis  was  once  constructed 
an  enormous  amount  of  theorization  as  to  ''uric-acid  diathesis," 
"uric-acid  intoxication, "  "lithemia, "  etc.,  until  it  came  to  be  popularly 
believed  that  a  large  share  of  the  minor  ailments  of  humanity,  and  in 
particular  all  non-infectious  diseases  of  the  joints  and  muscles,  are 
dependent  upon  the  presence  of  excessive  quantities  of  uric  acid  or 
urates  in  the  blood.  But  it  may  safely  be  stated  that  at  the  present 
time  there  exists  no  good  evidence  which  makes  it  probable  that  uric 
acid  is  responsible  for  any  pathological  conditions  whatever,  except 
uric-acid  calculi,  "uric-acid  infarcts"  in  the  kidneys,  and  certain 
manifestations  of  gout.  Uric  acid  is  possessed  of  but  a  very  shght 
degree  of  toxicity,  and  the  body  is  able  to  get  rid  of  it  in  such  large 

"  See  Siven,  Arch.  ges.  Physiol.,  1914  (157),  582;  Thannhauser  and  Dorf- 
muUor,  Zeit.  physiol.  Chem.,  1918  (102),  148. 

2"^  Review  on  uric  acid  metabolism  and  many  data  given  by  Host,  Nord.  Mag. 
Laev.,  1917  (78  Suppl.).     See  also  Jour.  Biol.  Chem.,  1919  (38),  17. 


DISTRIBUTION  OF  URIC  ACID  (iiio 

measure  that  an  actual  iMtoxifati(jn  witli  uric  acid  pn^hably  never 
occurs. 

The  amount  present  in  tlie  urine  nuiy  be  very  considerably  in- 
creased by  eating  food  ricli  in  jnirines,  of  wliich  sweet-breads,  liver, 
and  kidney  are  the  best  examples;  and  also  coffee  with  its  caffeine 
(trimethyl  purine),  may  give  rise  to  a  little  uric  acid,  although  the 
methylated  purines  seem  to  be  destroyed  in  large  part,  or  eliminated 
as  something  else  than  uric  acid.  Large  quantities  of  meat  will  also 
increase  the  uric  acid,  because  of  the  free  purines  contained  in  muscle; 
and  even  a  diet  rich  in  proteins  free  from  purine  will  also  increase  the 
uric  acid  excretion  over  that  of  a  low  protein  diet.-*  On  a  purine-free 
diet  the  excretion  of  endogenous  uric  acid  is  increased  by  increasing 
even  the  non-protein  calories  (Host).-^  However,  the  amount  of  uric 
acid  in  the  blood  is  not  correspondingly  raised  by  purine-rich  diets, ^^ 
this  being  regulated  by  the  binding  function  of  the  tissues  and  by  excre- 
tion through  the  kidneys.-^  According  to  Folin  and  Denis-^  human 
blood  normally  contains  1.5-2.5  mgs.  uric  acid  in  100  c.c,  and  the 
amount  bears  no  fixed  relation  to  the  amount  of  urea  and  total  non- 
protein nitrogen  of  the  blood. ^°  All  the  uric  acid  in  human  blood 
seems  to  exist  free  as  monosodium  urate,  and  not  in  a  colloidal  state 
(Gudzent).^^  Any  difficulty  in  renal  ehmination  is  usually  accom- 
panied by  an  increase  in  the  amount  of  uric  acid  in  the  blood,  in  ure- 
mia as  much  as  15  to  20  mg.  being  sometimes  found  per  100  c.c.'- 
In  early  interstitial  nephritis  there  may  be  4  to  8  mg.  of  uric  acid  per 
100  c.c.  blood  without  a  corresponding  increase  in  urea  and  creatinine, 
W'hich  suggests  that  uric  acid  may  be  less  easily  excreted  by  the  dis- 
eased kidney  than  the  other  chief  nitrogenous  constituents  of  the 
urine. 

In  normal  individuals  there  seems  to  be  httle  uric  acid  present  in 
the  tissues.  Bj^  using  Folin's  method,  Fine^^  found  that  various  tis- 
sues contain  quantities  comparable  to  that  in  the  blood  of  the  same 
person,  whether  this  is  normal  or  increased  in  amount.  Ordinarily 
these  quantities  are  not  sufficient  to  permit  readily  of  isolation  of  the 
uric  acid  in  a  pure  state,  but  in  the  tissues  of  a  young  woman  who  died 
after  complete  suppression  of  urine  for  nine  days  following  poisoning 
with  HgCl2,  I  found  considerable  amounts. ^^     Whenever  much  de- 

=«  Taylor  and  Rose,  Jour.  Biol.  Chem.,  1914  (18),  519;  Lewis  and  Doisv,  ibid., 
1918  (36),  1. 

2"  See  Denis,  Jour.  Biol.  Chem.,  1915  (23),  147. 

-^  Stocker  found  uric  acid  in  saliva,  increased  in  all  conditions  associated 
with  uricemia  (Inaug.  Dissert.,  Zurich,  1913). 

"Jour.  Biol.  Chem.,  1913  (14),  29;  Arch.  Int.  Med.,  1915  (16),  33. 

^0  In  infants  the  amount  is  sUghtlv  lower,  about  1.3  to  1.7  mg.  Liefraann, 
Zeit.  Kinderheilk.,  1915  (12),  227. 

"  Zeit.  klin.  Med..  1916  (82),  409. 

32  See  FoUn  and  Denis,  Arch.  Int.  Med.,  1915  (16),  33;  Meyers  and  Fine, 
ihid.,  1916  (17),  570;  Baumann  et  al,  ibid.,  1919  (24),  70. 

"  Jour.  Biol.  Chem.,  1915  (23),  473. 

"  Jour.  Biol.  Chem.,  1916  (26),  319. 


636  URIC-ACID  METABOLISM  AND  GOUT 

struction  of  the  nucleoproteins  of  the  tissues  is  occurring  in  the  body, 
the  ehmination  of  endogenous  uric  acid  becomes  abnormally  raised,  the 
best  examples  being  the  resolution  of  pneumonic  exudates,  and  leu- 
kemia, especially  leukemia  under  x-ray  treatment  (q.  v.) .  In  neither 
of  these  conditions,  however,  do  any  symptoms  or  tissue  changes  arise 
that  can  be  referred  to  the  excessive  uric  acid. 

GOUT 

Introducing  this  subject,  one  cannot  do  better  than  to  quote  v. 
Noorden's  statement  that  "It  is  not  to-day  very  alluring  to  write  any 
thing  regarding  the  theory  of  gout,  especially  in  a  book  which  is  essen- 
tially devoted  to  the  presentation  of  facts.  All  the  theories  advanced 
up  to  the  present  time  have  fared  badly.  The  positive  material  is 
much  too  insufficient  and  much  too  ambiguous."  After  adjusting 
the  many  contradictory  statements  of  earlier  investigators,  the  pres- 
ent status  of  our  conception  of  uric-acid  metabolism  in  gout  may  be 
briefly  summarized  as  follows:  The  excretion  of  uric  acid  in  patients 
with  chronic  gout,  when  kept  upon  a  definite  diet,  does  not  differ 
greatly  from  the  excretion  of  normal  individuals  on  the  same  diet. 
Normally  the  elimination  of  uric  acid  varies  within  rather  wide  limits, 
even  on  a  constant  diet,  but  the  excretion  in  chronic  gout  tends  to 
fall  at  or  slightly  below  the  lower  normal  limits.  As  a  rule,  gouty 
patients  on  a  purine-free  diet  excrete  less  endogenous  uric  acid  than 
normal  persons,  and  when  given  purines  in  the  food  the  rate  of  ex- 
cretion of  these  exogenous  purines  is  slower  than  normal. ^^  There 
seems  to  be  no  particular  relation  between  the  amount  of  uric  acid  in 
the  blood  and  the  occurrence  or  severity  of  attacks. ^"^  This  uric  acid 
is,  according  to  the  best  evidence,  in  a  free  state,  and  not  combined, 
as  was  at  one  time  urged  by  several  students  of  gout. 

Analyses  of  the  blood  in  120  cases  of  gout  by  Gettler  and  St. 
George^^  gave  the  following  figures,  in  mg.  per  100  c.c.  of  blood: 

Normal  Gout 

Nonprotein  nitrogen 25  to    40  30  to    55 

Urea  nitrogen 10  to     18  15  to    35 

Creatinine 0.1  to  0.8  1    to  2.8 

Uric  acid 0.5  to  3.0  1.5  to  8.5 

Sugar 00  to  110  85   to  140 

Alkali  reserve — percentage 53  to    SO  50  to    80 

^^  According  to  Gudzent's  studies  (Zcit.  physiol.  Chem.,  1909  (03),  455)  in 
nearly  all  cases  of  gout  the  blood  contains  as  nuich  or  more  inono-sodiuni  urate 
than  it  can  hold  in  solution  (8.3  ing.  per  100  c.c),  so  that  it  is  often  actually  a 
.supersaturated  solution  of  the  relatively  insoluble  lactim  form  of  urate.  Even  on 
a  purine-free  diet  the  blood  of  the  gouty  usually  contains  an  excess  of  uric  acid  (4 
to  9  ing.  per  100  c.c).  These  figures,  however,  are  much  higher  than  those  ob- 
tained by  more  modern  methods. 

="  Pratt,  Amcr.  Jour.  Med.  Sci.,  1910  (151),  92;  Bass  and  Herzberg,  Deut. 
Arch.  klin.  Med.,  1910  (119),  482. 

"  Jour.  Anier.  Med.  Assoc,  1918  (71),  2033. 


METABOLISM  IN  GOUT  <):i7 

Tlu'ir  deductions  arc  as  follows:  Cases  of  gout  as  a  Kciicral  rule 
show  some  increase  in  the  uric  acid  content  of  the  blood,  though  some 
of  the  chronic  cases  were  within  normal  limits  from  this  standpoint. 
The  increase  is  more  marked  in  the  acute  type  of  the  disease.  The 
uric  acid  content  of  the  hlood  in  cases  of  gout  is  abnormally  high  with- 
out a  corresponding  increase  in  the  nonprotein  nitrogen  products  of 
the  blood ;  but  the  majority  of  cases  show  a  slight  but  constant  increase 
in  the  nonprotein  nitrogen  and  creatinin.  An  increase  of  uric  acid  in 
the  blood,  with  the  patient  on  a  purin-free  diet,  may  be  a  symptom, 
but  is  not  diagnostic,  of  gout. 

McClure  and  Pratt^**  found  more  than  3  mg.  per  100  c.c.  in  their 
cases  when  on  purin-free  diet,  and  also  recognize  that  this  uricaemia 
is  not  diagnostic  of  gout.  The  former  reports  that  in  gout  there  is 
evidence  of  impaired  renal  function. ^^  If  abundant  purines  are  fed  to 
gouty  patients  they  may  be  found  to  have  less  than  normal  capacity 
to  excrete  the  excess/"  also  explainable  on  the  ground  of  renal  ineffici- 
ency. 

In  the  intervals  between  the  attacks  of  acute  gout  the  elimination 
of  uric  acid  is  said  to  remain  within  the  normal  limits;  however,  for  a 
period  of  one  to  three  da3's  before  each  acute  attack  the  amount  of  uric 
acid  is  usually  decreased  considerably.  With  the  onset  of  the  attack 
the  amount  of  uric  acid  excreted  becomes  increased,  and  for  a  few 
days  remains  above  the  average,  then  subsides  to  about  the  normal. 
Of  these  two  features,  the  increased  output  of  uric  acid  during  the 
attack  seems  to  be  more  constant  than  the  reduced  output  preceding 
it,  but  cases  occur  in  which  the  uric  acid  excretion  shows  no  variation 
from  that  of  normal  persons.  In  certain  cases  of  rheumatoid  arth- 
ritis the  behavior  of  the  purine  metabolism  resembles  that  of  gout.'*^ 

As  yet  we  have  no  definite  information  either  as  to  the  cause  of 
this  behavior  of  the  uric  acid  during  the  paroxyms  of  acute  gout, 
or  as  to  its  part  in  causing  the  paroxysm.  However,  in  view  of  the 
fact  that  monosodium  urate  is  found  in  the  joints  during  the  attacks, 
it  seems  most  probable  that  for  some  as  yet  unknown  reason  there 
occurs  a  precipitation  or  anchoring  of  the  urates  in  the  tissues,  which 
is  associated  with  the  attacks  of  pain  and  swelling.  We  do  not  know, 
however,  that  it  is  the  deposition  of  urates  that  causes  the  attacks. 
Indeed,  the  fact  that  uric-acid  retention  precedes  the  attack,  rather 
than  accompanies  it,  seems  to  suggest  that  it  is  the  absorption  of  the 
urate  rather  than  its  deposition  in  the  joints  that  is  responsible  for  the 
local  disturbances.  It  is  also  possible  that  during  the  period  of  re- 
tention the  uric  acid  is  held  in  the  blood  in  some  form  that  cannot 
be  eliminated  by  the  kidnej^  and  that  its  deposition  in  the  joints  in 

S8  Arch.  Int.  Med.,  1917  (20),  481— bibliography. 
39  Ibid.,  1917  (20),  641. 

«  Rosenbloom,  Jour.  Amer.  Med.  Assoc,  1918  (70)  285. 

«  W.  J.  Mallorj',  Jour.  Path,  and  Bact.,  1910  (15),  207;  Ljungdahl.  Zeit,  klin. 
Med.,  1914  (49),  177. 


638  URIC-ACID  METABOLISM  AND  GOUT 

an  absorbable  form  occurs  simultaneously  with  the  attack.  The  fail- 
ure of  recent  studies  on  the  enzymatic  transformation  of  purines  to 
locate  anywhere  in  the  human  body  an  enzyme  destroying  uric  acid, 
makes  hazardous  the  attempt  to  explain  gouty  metabohsm  as  a  result  of 
enzymatic  abnormalities.  However,  there  can  be  little  doubt  that  the 
fundamental  reason  for  the  existence  of  uric  acid  gout  in  man  lies  in  the 
inabilityof  the  human  organism  to  destroy  uric  acid.  Because  man  can- 
not destroy  uric  acid  rapidly  by  oxidation,  as  can  all  other  mammals,  he 
is  always  a  potential  victim  of  uric  acid  retention  and  deposition. 

It  should  be  mentioned  in  addition  that  it  is  not  the  uric-acid 
metabohsm  alone  that  is  altered  in  gout.  Irregular  periods  of  nitro- 
gen retention  and  nitrogen  loss  are  quite  constant  features.  The 
cause  of  this  variability,  and  the  form  in  which  the  nitrogen  is  re- 
tained, are  quite  unknown,  although  there  is  some  evidence  that  the 
retained  nitrogen  is  in  the  form  of  purine  bodies  (Vogt).  ]\Iost  of 
the  excessive  loss  occurs  during  the  acute  attacks,"*-  and  the  retention 
of  nitrogen  between  attacks  may  be  partly  to  repair  the  loss;  against 
this,  however,  is  the  fact  that  there  is  not  sufficient  gain  in  weight 
to  account  for  all  of  the  nitrogen  retention.  Associated  with  the  de- 
layed excretion  of  ingested  purines  is  also  a  delayed  excretion  of  the 
other  nitrogenous  products  of  protein  food.^^  The  proportion  of 
purine  bases  to  uric  acid,  is  not  altered  in  gouty  urine. ^^^  The  state- 
ments in  regard  to  phosphoric  acid  elimination,  which  depends  largely 
on  decomposition  of  nucleins,  are  contradictory,  but  it  seems  probable 
that  it  shows  no  characteristic  alterations  in  gout.  Amino  acids,  espe- 
cially glycine,  are  said  to  be  excreted  in  excess. "^^  There  is  no  significant 
change  in  the  basal  metabolism.'*^ 

It  may  be  seen  from  the  foregoing  discussion  that  we  neither  under- 
stand fully  the  intricacies  of  metabolism  in  gout,  nor  know  whether 
uric  acid  is  responsible  for  either  the  acute  painful  attacks  or  for 
the  anatomical  alterations  in  the  kidneys,  heart,  and  blood  vessels. 
Indeed,  Daniels,  and  McCrudden'*^  have  shown  that  it  is  possible  for 
gouty  patients  to  have  a  persistently  low  content  of  uric  acid  in  the 
blood,  below  the  average  normal  quantity,  and  to  have  typical  acute' 
attacks  without  change  in  either  the  uric  acid  content  of  the  blood  or 
its  excretion;  attacks  were  even  observed  to  occur  Avhen  the  blood  uric 
acid  was  at  a  subnormal  figure  from  administration  of  atophan,  which 
increases  its  elimination.  Furthermore,  Bass  and  Horz]')erg"'**  found 
that  uric  acid  can  be  injected  into  the  blood  of  gouty  subjects  until  the 
blood  contains  as  much  as  10  mg.  per  100  c.c.  without  causing  any 
joint  symptoms. 

«  BruKsch,  Zeit.  exp.  Path.  a.  Tlior.,  1906  (2),  610. 

"Leveneand  Kristeller,  Jour.  Exj).  Mod.,  1912  (16),  303. 

"Hefftor,  Dent.  Arch.  klin.  Med.,  1913  (109),  322. 

"  Bih-ffor  and  Schwerinor,  Arch.  exp.  Patli.,  1913  (74),  353. 

•'«  Wentworlli  and  McClure,  Arcli.  Int.  Med.,  1918  (21),  84. 

"  Arch.  Int.  Med.,  1915  (15),  1046. 

"  Deut.  Arch.  klin.  Med.,  1916  (119),  482. 


URATE  DEPOSITS  639 

It  is  very  possible  that  some  entirely  different  product  of  metabo- 
lism than  uric  acid  is  ros|)onsil)lo  for  most  of  the  chanpos  and  symi)- 
toms  of  gout"'^ — -indeed,  this  would  seem  to  be  the  case  were  it  not  for 
the  great  frequency  of  the  deposition  of  monosodium  urate  in  the 
joints  and  cartilages,  both  during  the  acute  attacks  and  in  chronic 
gout.  This  indicates  that  there  is  surely  something  abnormal  in  the 
conditions  of  uric-acid  solution  and  circulation.  Why  the  urate  is 
precipitated  in  these  definite  places  is  another  of  the  many  unsolved 
problems  of  gout.  The  local  nature  of  the  depo.sition  indicates  that  it 
must  depend  upon  local  changes;  but  the  hypothesis  that  there  occur 
first  degenerative  changes  in  the  tissues  which  determine  the  precipi- 
tation of  the  urate,  seems  to  have  been  disproved  by  the  demonstra- 
tion that  the  deposition  of  the  urates  precedes  the  necrosis.  The 
fact  that  the  presence  of  other  sodium  salts  in  a  solution  decreases 
the  solubility  of  urates  in  that  solution,  and  the  fact  that  cartilage 
and  tendons  are  richer  in  sodium  salts  than  the  blood,  may  possibly 
have  something  to  do  with  the  fact  that  the  urates  are  precipitated  in 
these  particular  tissues.  On  the  other  hand  is  the  fact  that  in  leu- 
kemia and  nephritis  we  msLy  have  a  higher  concentration  of  uric  acid 
in  the  blood  than  in  gout,  and  this  uricsemia  may  be  protracted,  with- 
out gouty  deposits  or  joint  symptoms.  Bass  and  Herzberg'''*  found 
that  the  uric  acid  content  of  the  joint  fluid  was  approximately  the 
same  as  that  of  the  blood  in  patients  without  gout,  although  in  two 
gouty  uremics  they  found  18.5  and  20.8  mg.  in  the  joint  fluid  with 
onl}^  10  and  8.2  mg.  in  the  blood.  Thej^  also  found  that  intravenous 
uric  acid  injection  caused  less  uricsemia  in  the  goutj^  in  spite  of  re- 
duced renal  excretion,  and  hence  they  conclude  that  in  gout  the  tissues 
have  an  increased  capacity  for  taking  up  uric  acid. 

The  histology  of  urate  deposits,  both  experimental  and  gouty,  has  been  care- 
fuUj'  studied  by  Freudweiler,*"  His,»'  Krause,"^  and  Rosenbach.*^  Their  results 
all  indicate  that  uric  acid  and  urates  excite  some  slight  inflamniator\'  reaction, 
cause  a  slight  local  necrosis,  and  seem  to  act  as  a  weak  tissue  poison  (His).  How- 
ever, they  may  be  deposited  without  causing  necrosis  (Rosenbach).  Possibly 
part  ot  the  material  observed  in  areas  of  urate  deposition,  and  generally  considered 
as  necrotic  tissue,  merely  represents  the  framework  of  the  crystalline  deposit 
(Krause).  When  experimentally  injected,  the  urates  are  absorbed  slowly  by 
phagocytic  leucocytes  and  giant -cells.  Why  the  gouty  tophi  can  be  deposited  in 
the  chronic  process  and  cause  no  pain  or  inflammation,  while  in  acute  gout  de- 
position of  urates  seems  to  cause  such  marked  symptoms,  is  also  an  unanswered 
question,  unless  we  accept  the  explanation  that  the  slower  rate  of  deposition  and 
the  lack  of  dissolved  urates  account  for  the  absence  of  symptoms  with  the  tophi. *^ 
Magnus-Levy  holds  with  Pfeiffer,  that  the  local  inflammatory  processes  must  be 
ascribed  to  dissolved  urates,  since  they  often  extend  for  some  distance  about  the 
joints,  and  hence  the  attack  is  ascribable  to  the  solution  rather  than  the  fornia- 

*^  In  swine  a  "guanine  gout"  occurs;  see  Schittenhelm  and  Bendix,  Zeit. 
phvsiol.  Chem.,  1906  (48),  140. 

"=0  Deut.  Arch.  klin.  ISIed.,  1899  (63),  266. 

^^  Ibid.,  1900  (67),  81. 

"  Zeit.  klin.  Med.,  1903  (50),  136. 

5-^  Virchow's  Arch.,  1905  (179),  359. 

^*  Almagia  (Hofmeister's  Beitr.,  1905  (7),  466)  has  found  that  joint  cartilage 
placed  in  urate  solutions  becomes  filled  with  cr^-stals.  which  infiltration  does  not 
occur  with  cartilage  of  any  other  origin,  or  with  tendons. 


640  URIC-ACID  METABOLISM  AND  GOVT 

tion  of  the  deposits,  a  fact  in  harmony  with  the  known  increased  ehmination  of 
uric  acid  during  the  attack. 

That  urates  may  cause  necrosis  of  the  tissues  has  been  definitely  estabUshed, 
and  this  may  lead  to  connective-tissue  formation  and  contraction.^^  But  the 
actual  increase  of  uric  acid  in  the  blood  and  tissues  in  gout  is  so  slight  that  we  are 
not  warranted  in  saying  that  the  usual  tendency  to  sclerosis  in  all  the  organs  in 
gout  is  due  to  the  action  of  uric  acid,  rather  than  to  some  other  unknown  agent 
or  agents.  Excess  of  uric  acid  in  the  blood  is  by  no  means  pathognomonic  of 
gout,  for  we  may  have  relatively  great  excesses  of  uric  acid  in  the  blood  in  leuke- 
mia, in  some  cases  of  nephritis,  and  after  eating  large  amounts  of  nucleoproteins, 
without  a  symptom  of  gout.  Furthermore,  it  is  quite  possible  that  the  precursors 
of  uric  acid,  the  purine  bases,  are  responsible  for  more  harm  than  the  uric  acid  it- 
self. Thus,  administration  of  adenine  to  dogs  and  rabbits  will  produce  degenera- 
tive changes  in  the  kidneys,  associated  with  the  deposition  of  substances  resembling 
uric  acid  and  urates  in  the  renal  tissue;  and  MandeP^  states  that  purine  bases 
may  cause  fever,  independent  of  inlection. 

Many  have  looked  upon  renal  alterations,  leading  to  failure  of 
excretion  of  uric  acid,  as  the  primary  cause  of  gout;  but  the  evidence 
in  favor  of  this  is  faulty,  because  frequently  renal  changes  are  slight 
or  entirely  absent  in  gout,  whereas  marked  nephritis  of  all  forms  may 
exist  without  the  coexistence  of  gout,  and,  as  mentioned  above,  the 
kidney  in  gout  may  show  no  lack  of  ability  to  excrete  uric  acid  injected 
into  the  tissues.  Magnus-Levy,  however,  seems  to  believe  that  a 
renal  retention  of  uric  acid  is  of  importance,  and  that  it  may  occur  with- 
out morphological  changes  in  the  kidneys.  The  newer  methods  of 
blood  analysis  (Folin)  have  given  support  to  this  view,  and,  as  pointed 
out  in  preceding  pages, Fine^^  and  numerous  others  have  called  attention 
to  the  fact  that  in  early  interstitial  nephritis  the  blood  shows  a  greater 
increase  in  uric  acid  than  in  urea  or  <?reatinine,  as  if  the  diseased  kidney 
found  more  difficulty  in  excreting  uric  acid  than  the  other  substances. ^^ 
As  a  result  the  blood  in  early  nephritis  may  show  quite  the  same  fig- 
ures for  uric  acid,  urea  and  creatinine  as  are  found  characteristically 
in  gout.  Although  in  some  cases  one  finds  normal  amounts  of  uric 
acid  in  the  blood  in  gout,  this  seems  to  be  exceptional.  Hence  we 
are  still  confronted  with  the  question  whether  gout  is  anything  more 
than  a  form  of  nephritis  in  which  chiefly  uric  acid  excretion  is  impaired 
or  whether  there  does  exist  a  special  disease,  gout,  which  causes 
uric-acidemia   more  or  less  independently  of  renal  abnormalities.*^ 

URIC-ACID  INFARCTS^" 

Uric-acid  infarcts,  as  the  deposits  of  urates  and  uric  acid  observed  in  the 
kidneys  of  at  least  half  of  all  children  dying  within  the  first  two  weeks  of  life  are 
called,  give  evidence  of  the  slightness  of  the  toxic  effects  of  these  substances  upon 
the  tissues.     Usually  little  or  no  change  occuri-  in  the  renal  tubules  as  a  result  of 

^^  Because  the  gouty  tophi  do  not  suppurate,  even  when  ulcerated  through  the 
skin,  it  has  been  suggested  that  the  urates  have  antiseptic  properties.  Bcudix 
(Zeit.  klin.  Med.,  1902  (44),  165),  however,  could  not  demonstrate  such  antiseptic 
properties  experimentally.  Not  always  do  the  tophi  consist  solely  or  even  largely 
of  urates,  but  these  may  be  replaced  by  calcium  salts  (Kahn,  Arch.  Int.  Med., 
1913  (11),  92). 

<*«  Amcr.  Jour.  Physiol.,  1904  (10),  452;  1907  (20),  439. 

"  Jour.  Amer.  Med.  Assoc,  1916  (66),  2051. 

"  See  also  Denis,  Jour.  Biol.  Chem.,  1915  (23),  147. 

'"'  See  McClure,  Arch.  Int.  Med.,  1917  (20),  641. 

60  See  discussion  by  Wells  and  Corper,  Jour.  Biol.  Chem.,  1909  (6),  321. 


UniC  ACID  IXFAh'CTS  r,4i 

these  depositions,  except  such  as  can  be  attril)iiled  to  their  iiieolianicul  effect/' 
but  they  may  serve  as  the  starting  jwint  of  calcuU.  The  rejison  for  tlie  formation 
of  these  infarcts  is  not  at  all  understood.  Sjjienelljerg"  found  it  iMjssible  to  cause 
them  experimentally  in  young  dogs,  in  which  they  do  not  occur  naturally,  by  injec- 
tion of  0.25  gram  ol  uric  acid  per  kilo.  He  was  unable  to  explain  why  thi.s  deposi- 
tion should  occur  in  young  animals  but  not  in  old,  for  he  could  not  find  evidence  of 
lessened  oxidative  power  on  the  part  of  young  animals,  and  the  solvent  fjower  of 
infants'  urine  was  found  equal  to  or  greater  than  that  of  adults.  Other  authors 
however,  have  found  a  lower  oxidative  power  in  young  aninuds,  and  Mendel  and 
Mitchell"  have  found  that  in  the  embryo  pig  uricolytic  enzyme.^,  do  not  appear  un- 
til just  at  or  just  after  the  time  of  birth.  As  human  tissues  have  no  demonstrable 
I)ower  to  oxidize  uric  acid,  however,  these  animal  experiments  cannot  be  applied  to 
the  \iric  acid  infarcts  in  human  infants.  Possibly  the  uric-acid  infarcts  of  infants 
are  the  result  of  the  great  destruction  of  nucleoproteins  that  results  from  the 
change  of  the  nucleated  fetal  red  corpuscles  to  the  non-nucleated  adult  form, 
or  from  a  destruction  of  leucocytes  which  is  said  to  take  place  at  the  time  of  birth! 
Flensberg  believes  that  a  hyaline  substance  is  secreted  in  the  urine  of  'new-born 
infants  which  acts  as  a  matrix  for  urate  deposition.  McCruddcn  considers  the 
high  concentration  of  infants'  urine  an  important  factor.  Minkowski'^  observed 
that  administration  of  adenine  to  dogs  led  to  a  deposition  of  uric  acid  or  some 
similar  substance  in  the  kidneys.*^"  Schittenhelm"  found  the  same  deposits  in  the 
kidneys  of  rabbits  fed  adenine,  but  not  when  they  were  fed  guanine.  According  to 
Nicolaier,"  the  crystals  thus  deposited  are  not  uric  acid  or  urates,  but  6-amino-2-8- 
dioxypurine,  derived  from  the  adenine  (6-amino-purine)  by  direct  but  incomplete 
oxidation.  He  could  not  find  this  substance  in  either  human  urine  or  in  a  uric- 
acid  calculus.  Eckert''^  obtained  urate  deposits  by  intravenous  injection  into 
rabbits  of  at  least  0.08  gm.  per  kilo,  but  subcutaneous  injections  were  ineffective; 
injury  to  the  renal  epithelium  by  whatever  cause  interferes  with  this  deposition  of 
urates.  These  experimental  irifarctions  are  undoubtedly  related  to  the  human 
form,  and  indicate  that  the  latter  depend  upon  the  presence  of  an  excessive  amount 
of  uric  acid  in  the  infants'  urine,  in  which  a  ratio  of  uric  acid  to  urea  of  7.9  to  74.9, 
as  against  the  adult  ratio  of  about  2  to  85,  was  found  by  Sjoquist.  According  to 
Niemann,^'  in  the  first  few  days  of  life  the  infant  excretes  from  80  to  100  mg.  of 
uric  acid  daily,  while  after  the  fifth  day  the  amount  falls  to  30  to  40  mg.  daily. 
Similar  figures  were  obtained  by  Schloss  and  Crawford,^"  who  also  found  a  corre- 
sponding increase  in  the  phosphoric  acid,  showing  that  the  uric  acid  must  originate 
from  nucleoproteins.  The  blood  of  fetus  and  mother  have  the  same  uric  acid 
content,^"  but  after  birth  the  infant's  blood  has  more  during  the  first  three  or  four 
days,  paralleling  the  high  excretion." 

Adult  kidneys  may  also  show  uric  acid  deposits  in  the  tubules  of  the  papilla*, 
independent  of  gout.  They  occur  as  a  result  of  cell  decomposition,  according  to 
M.  B.  Schmidt,^^  who  found  them  especially  in  pneumonia,  leukemia  and  sarcoma, 
but  not  in  carcinoma. 

^^  I  have  observed  a  case  of  fatal  hematuria  neonatorum,  associated  with  most 
extensive  hemorrhagic  infarction  of  both  kidneys.  In  the  bloody  urine  B.  coli 
was  found  in  l^rge  numbers.  From  the  anatomical  findings  and  history  it  seemed 
quite  possible  that  the  injury  of  the  kidneys  by  uric-acid  infarcts  might  have 
determined  the  localization  of  the  bacteria  in  these  organs,  with  resulting  hemor- 
rhages.    (Trans.  Chicago  Path.  Soc,  1909  (7),  242.) 

«2  Arch.  exp.  Path.  u.  Pharm.,  1898  (41),  428. 

"  Amer.  Jour.  Physiol,  1907  (20),  97. 

"Arch.  exp.  Path.  u.  Pharm.,  1898  (41),  375. 

""  Abderhalden  and  Kankeleit  (Zeit.  exp.  Med.,  1916  (5),  172),  have  produced 
renal  deposits  by  feeding  large  amounts  of  tyrosine,  glycine  and  leucineimid  to 
rabbits.  These  deposits  consisted  of  the  amino  acid  as  fed,  and  caused  suppres- 
sion of  urine  by  blocking  up  the  tubules.  They  also  caused  necrosis  and  inflamma- 
tory reactions. 

«  Ibid.,  1902  (47),  432. 

««  Zeit.  klin.  Med.,  1902  (45),  359. 

«'  Arch.  exp.  Path.,  u.  Pharm.,  1913  (74),  244. 

"  Jahrb.  f.  Kinderheilk.,  1910  (71),  286. 

"Amer.  Jour.  Dis.  Children,  1911  (1),  203. 

'»  Slemons  and  Bogert,  Jour.  Biol.  Chem.,  1917  (82),  63. 

""■  Kingsbury  and  Sedgwick,  ibid.,  1917  (31),  261. 

"2  Verb.  deut.  Path.  Ges.,  1913  (16).  329. 


CHAPTER  XXIV 
DIABETES 

By  R.  T.  Woodyatt 

Introduction. — As  with  gout  and  the  problems  of  purine  metabo- 
hsm,  so  with  diabetes  a  vast  amount  of  study  has  been  expended  be- 
cause of  the  integral  connection  of  this  disease  with  broader  problems 
of  physiology,  and  in  particular  with  the  metabolism  of  the  carbo- 
hydrates and  the  fats,  the  nature  of  internal  secretions,  and  the  func- 
tion of  the  kidneys.  It  is  impossible  in  this  place  to  review  the  entire 
literature  and  history  of  the  subject,  a  key  to  which  will  be  found  in 
the  works  of  the  writers  cited  below.  ^  This  chapter  will  be  devoted 
only  to  an  outline  of  the  chief  established  facts,  with  an  indication  of 
the  main  hnes  along  which  the  thought  of  leading  students  has  been 
directed.  It  will  involve  a  brief  discussion  of  the  problems  of  carbo- 
hydrate physiology,  but  only  in  so  far  as  they  are  contingent  upon  the 
main  topic — while  for  a  discussion  of  that  anomaly  of  the  metabolism 

1  Older  Ldterature: 
Bouchardat — De  la  glycosurie  ou  diabete  sucrc,  Paris,  1875. 
Kiilz — -Beitrage  zur  Path,  und  Ther.  der  Diabetes  Melitus,  Marburg,  1874- 

5. 
Bernard — Legons  sur  le  Diabete  et  la  Glycogenese  Animale,  Paris,   1877; 

Vorlesungen  iiber  Diabetes,  Berlin,  1878. 
Cantani — Diabetes  Melitus  (German  translation  by  Kahn),  Berlin,  1880. 
Frerichs — Ueber  den  Diabetes,  Berlin,  18S-4. 
Larger  Works: 

Naunyn — Der  Diabetes  Melitus,  Berlin,  1906.     Diabetes  Melitus;  in  Noth- 

nagel's  Handbuch  (2nd),  Vienna,  1906. 
Lepine — Le  diabete  sucre,  Paris,  1909. 
von    Noorden — (a)    Die   Zuckerkrankheit    (6th),    Berlin,    1912.     (b)    New 

Aspects  of  Diabetes,  New  York,  1913. 
Pavy — Carbohydrate  Metabolism  and  Diabetes,  London,  1906. 
McLeod — Diabetes  (Longmans,  Green),  1918. 

Cammidge — Glycosuria  and  Allied  Conditions.      (Longmans,  Green),  1913. 
Allen — Glycosuria  and  Diabetes,  Boston,  1913. 
Foster — Diabetes  Melitus,  Philadelphia,  1915. 
Joslin — Treatment  of  Diabetes  Melitus,  New  York,  3rd.  Ed.,  1919. 
Monographs,  etc.: 

Magnus-Levj^ — Diabetes  Melitus;  in  Kraus  and  Brugsch,  Spezielle  Path.  u. 

Ther.  innerer  Krankheiten,  Berlin,  1913. 
Gigon — Neuere   Diabetes   Forschungen,   Ergebnisse  der  inneren   Medizin, 

1912,  IX,  p.  206. 
von  Mering — Behandlung  der  Diabetes  melitus;  in  Penzoldt  and  Stinzing's 

Handbuch  der  Spezielle  Therapie  (2nd),  1912. 
Lusk — Elements  of  the  Science  of  Nutrition  (3rd),  New  York,  1917. 
Benedict   and    Joslin — Metabolism  in  Diabetes  Melitus,  Carnegie  Institu- 
tion, Washington,  1910. 
Benedict  and  Joslin — A  Study  of  Metabolism  in  Severe  Diabetes,  ibid.,  1912. 
Allen,  Stillman  and  Fitz — Total  Dietary  Regulation  in  the  Treatment  of 
Diabetes.     Monograph  of  the  Rockefeller  Institute  for  Medical  Research, 
New  York,  1919. 

642 


GLYCOSUlilA  043 

whicli  leads  to  the  excretion  of  acetone,  accto-acotic  acid  and  ^-liy- 
droxybutyric  acid  in  the  urine,  the  reader  is  refernMl  to  the  section 
on  acidosis.     (Chapter  xx). 

Whereas  the  normal  urine  at  all  times  contains  reducing  substances 
and  substances  which  are  optically  active,  which  yield  crystalline 
compounds  with  the  hydrazines  and  respond  to  other  so-called  sugar 
tests,  these  substances  are  not  all  sugars,  nor  are  all  the  sugars  glucose. 
The  quantity  of  fermentable  reducing  substance  in  normal  urine 
averages  about  4  parts  in  10,000  (0.04  per  cent.)  acording  to  Lavesson.^ 
If  the  total  quantity  of  urine  were  1500  c.c.  this  would  imply  a  daily 
excretion  of  about  0.6  gm.  Bang  and  Bohmannson'  estimated  the 
total  reducing  substance  in  the  urine  of  normal  adults  as  between 
0.21  and  0.24  per  cent.,  of  which  about  18  per  cent,  was  fermentable 
(0.038  to  0.043  per  cent,  of  fermentable  reducing  substances  in  the 
urine).  This  is  doutbless  subject  to  change  depending  on  the  diet 
and  other  factors.  Benedict,  Osterberg  and  Neuwirth'*  found  an 
excretion  of  fermentable  reducing  substance  ranging  between  0.903 
and  1.161  gm.  in  twenty-four  hours  in  the  case  of  a  normal  adult  on 
an  ordinary  mixed  diet.  During  a  fast  it  fell  to  zero,  according  to 
these  writers,  and  on  a  diet  low  in  carbohj-'drate  it  averaged  0.75  gm. 
while  with  a  high  carbohj^drate  diet  it  rose  to  1.5  gm. 

When  an  abnormal  amount  of  sugar  occurs  in  the  urine,  regardless 
of  the  kind,  the  condition  may  be  called,  in  accordance  with  Naunyn's 
suggestion,  melituria.  When  the  sugar  is  glucose  (dextrose),  the 
term  glycosuria  is  applied;  when  levulose,  levulosiiria ,  and  so  on. 
Other  known  forms  of  melituria  are  lactosuria,  galactosuria,  fructo- 
suria,  pentosuria,  etc.  All  these  are  but  symptoms,  manj'  of  them 
being  caused  by  a  variety  of  mechanisms,  which  will  be  discussed 
presently. 

The  term  diabetes  is  often  loosely  used  to  cover  any  variety  of 
melituria,  but  is  is  preferably  limited  to  certain  forms;  namely,  to 
the  glycosurias  (or  the  mixed  meliturias  in  wliich  d-glucose  is  the 
predominating  sugar),  and  further  than  this,  to  those  particular 
glycosurias  which  continue  even  after  the  glycogen  reserves  of  the 
body  have  become  depleted  and  when  the  diet  is  free  of  carbohydrate; 
or,  to  those  transient  glycosurias  whose  nature  by  one  means  or  another 
can  be  proved  to  be  identical  with  the  continuous  forms  (latent  or 
mild  diabetes).  Over  against  these  are  the  meliturias  in  which  other 
sugars  than  glucose  play  the  chief  role,  and  glycosiirias  which  are  essen- 
tially transient  because  they  depend  solely  on  the  ingestion  or  admin- 
istration of  excessive  quantities  of  glucose  or  the  sudden  liberation 
into  the  blood  of  glucose  derived  from  preformed  glycogen  or  other 
fixed  compound  of  sugar. 

2  Biochem.  Zeit.,  1907  (4).  40. 

3  Zeit.  physiol.  Chem.,  1909  (63),  443. 
*  Jour.  Biol.  Chem.,  1918  (34),  217. 


644  DIABETES 

Thus,  the  glycosuria  which  follows  puncture  of  the  floor  of  the 
-fourth  ventricle  (Claude  Bernard's  piqure)  does  not  occur  in  animals 
which  contain  little  glycogen.  The  same  applies  to  the  adrenal, 
thyroid  and  hypophysis  glycosurias.  But  after  complete  pancreas 
extirpation  (pancreas  diabetes)  and  in  the  spontaneous  human  disease 
(diabetes  melitus)  or  its  counterpart  in  animals,  and  during  the  con- 
tinuous administration  of  phlorhizin,  the  glycogen  may  be  nearly  or 
quite  exhausted  and  the  diet  consist  solely  of  meat  and  fat  and  still 
the  glycosuria  will  continue.  On  the  other  hand  a  partial  pancreas 
extirpation,  a  mild  diabetes  melitus,  or  an  interrupted  phlorhiziniza- 
tion  may  give  rise  to  transient  glycosuria,  the  diagnosis  of  which  may  be 
difficult.  In  general,  experience  teaches  that  all  persistent  glycosurias 
prove  to  be  diabetic  and  that,  except  in  phlorhizin  poisoning,  ever}- 
genuine  diabetes  implies  a  disturbed  function  of  the  pancreas.  In 
forming  a  judgment  of  the  value  of  any  experimental  work  on  diabetes 
(histological,  chemical  or  clinical),  the  student  will  do  well  to  examine 
critically  the  records  of  quantitative  food  and  urinary  analyses  offered 
by  the  investigator,  to  show  what  type  and  what  grade  of  diabetes 
is  under  consideration. 

CARBOHYDRATE  PHYSIOLOGY 

Certain  facts  concerning  the  physiology  of  the  carbohydrates  may 
be  briefly  recalled  before  entering  into  the  discussion  of  the  individual 
meliturias. 

The  appearance  of  sugar  in  the  urine  implies  a  source  or  sources 
of  sugar  and  the  existence  of  a  kidney  membrane  of  such  a  physical 
character  that  molecules  of  sugar  may  migrate  through  it  with  a 
certain  degree  of  facility.  The  factors  which  may  influence  the  purely 
physical  penetrability  of  the  kidney  membrane  to  sugar  molecules  are 
those  involved  in  a  discussion  of  kidney  function  and  secretion  in 
general  and  need  not  be  elaborated  here. 

Assuming  that  the  physical  penetrability  of  the  kidney  membrane 
to  sugar  molecules  is  normal  and  that  it  varies  only  within  constant 
limits,  there  are  then  two  basic  moments  which  determine  how  much 
sugar  will  pass  into  the  urine.  These  are:  1.  The  rate  at  which  sugar 
molecules  enter  the  cells  constituting  the  kidney  membrame.  2.  The 
rate  at  which  these  molecules  of  sugar  undergo  chemical  change  into 
something  else  within  the  membrane. 

These  same  factors  of  supply  and  utilization  determine  the  elimina- 
tion of  sugar  from  any  cell  or  tissue  or  the  organism  as  a  whole,  but 
in  the  case  of  internally  situated  cells  the  elimination  is  directly  or 
indirectly  into  the  blood,  whereas,  in  the  case  of  the  kidney  cells 
sugar  may  pass  into  the  urine  as  well  as  into  the  blood  and  thus  leave 
the  body  permanently. 

Sugar  may  pass  out  of  a  cell  unchanged  when  the  rate  at  which 
it  enters  the  cell  (from  internal  and  external  sources)  exceeds  the  rate 


THE  EXCRETION  OF  SUGAR  645 

at  which  it  undergoes  tluingc  into  something  else  within  the  cell,  the 
same  holding  in  the  case  of  the  cells  forming  the  kidney  membrane. 
Thus  sugar  passes  from  the  kidney  membrane  into  the  urine  irhen.  the 
rate  at  which  sugar  molecules  enter  the  kidney  membrane  exceeds  the 
rate  at  which  they  are  denatured  or  utilized  within  it.  In  order  to  under- 
stand the  various  ways  in  which  melituria  may  be  produced  it  is 
necessary  to  analyze  further  these  factors  of  supply  and  utilization. 

The  Sugar  Supply  to  the  Kidneys  is  cliiefly  via  tho  hlooci,  althouRh  some  may 
be  liberated  within  the  kidney  cells  themselves  from  glycogen  deposits  or  from  the 
transformation  of  protein  during  metabolism.  The  factors  which  infliienre  the 
rate  at  which  sugar  molecules  enter  the  kidney  membrane  from  the  blood,  are 
immediate  and  remote. 

The  more  remote  factors  are  those  which  determine  the  quantity  of  sugar  in 
general  circulation,  and  the  distribution  of  blood  to  the  kidneys.  The  relative 
quantity  of  sugar  circulating  in  the  blood  will  depend  on  the  combined  rates  at 
which  sugar  enters  the  blood  from  the  outside  and  from  all  the  organs,  and  on  the 
rate  at  which  sugar  passes  out  of  the  blood  into  the  cells.  The  absolute  value  of 
these  factors,  supply  and  depletion,  and  their  ratio,  will  determine  fluctuations  of 
the  total  blood  sugar.  The  supply  to  the  kidneys  is  accordingly  influenced  by  the 
balance  of  supply  and  depletion  elsewhere. 

The  immediate  factors  which  determine  the  rate  at  which  sugar  is  brought  to  the 
kidney  membrane  will  include  all  of  those  which  may  influence  the  state  of  the 
blood  as  to  viscosity  etc.,  and  the  rate  at  which  sugar  actually  enters  the  kidney 
membrane  will  be  influenced  by  changes  in  the  state  of  that  membrane,  but  apart 
from  those  things  there  are  two  factors  of  major  importance  in  determining 
the  supply  of  sugar  to  the  kidney  membrane  at  any  instant,  namely,  the  concentrn- 
tion  of  sugar  in  the  blood  plasma  of  the  kidney  capillaries  and  the  extent  of  the  surface 
of  contact  between  the  blood  plasma  and  the  kidney  membrane.  In  accordance  with  a 
method  followed  by  M.  H.  Fischer  it  is  convenient  to  think  of  the  kidney  membrane 
simply  as  including  all  that  lies  between  the  blood  plasma  on  the  one  hand  and  the 
urine  on  the  other.  So  considered,  the  surface  of  contact  between  the  blood  plasma 
and  the  kidney  membrane  is  the  internal  surface  of  all  the  capillaries  of  the  cortex, 
in  so  far  as  these  are  filled  with  circulating  blood.  The  kidney  membrane  might  be 
conceived  otherwise  and  be  made  to  include  only  the  layer  of  epithelial  cells,  in 
which  case  the  surface  of  contact  might  be  considered  as  the  surface  of  those  cells 
in  so  far  as  they  are  bathed  in  plasma;  or  the  matter  might  be  carried  within  the 
cells,  in  which  case  the  problem  of  surface  would  become  one  of  surfaces  between 
phases  of  an  heterogeneous  system  in  the  chemical  sense,  and  so  on. 

For  present  purposes  it  is  convenient  to  mass  all  such  intermediate  factors  and 
deal  with  the  sum  of  their  effects,  i.  e.,  to  think  of  this  surface  as  the  internal 
surface  of  the  active  capillaries.  The  surface  of  contact  between  the  blood 
plasma  and  kidney  membrane,  so  considered,  is  clearly  subject  to  variations,  for 
as  more  or  less  blood  is  forced  into  the  capillaries  of  the  cortex  there  must  be  changes 
in  the  diameter,  length  or  number  of  capillaries  containing  circulatory  blood,  or  in 
all  three.  Any  or  all  of  these  changes  implj'  changes  of  capillary  surface.  It 
therefore  becomes  apparent  that  other  factors  remaining  the  same,  changing  vol- 
umes of  blood  in  the  cortical  capillaries  would  imply  changing  rates  of  sugar 
diffusion  from  the  blood  into  the  kidney  cells,  even  though  the  concentration  of 
sugar  in  the  blood  should  remain  unchanged.  And  it  is  also  apparent  that  if  the 
blood  should  be  diluted  with  water  in  such  a  way  as  to  double  its  volume  and  halve 
the  blood  sugar  percentage  this  would  not  of  necessity  change  the  rate  at  which 
sugar  was  passing  from  the  blood  into  the  kidney  membrane,  provided  the  extra 
volume  of  blood  developed  for  itself  a  proportional  amount  of  extra  capillary 
surface.'"'     Thus  it  has  been  shown  by  Epstein*  that  the  rate  at  which  sugar  is 

*»  This  raises  the  question  of  the  geometrical  form  of  capillaries  and  the  mechan- 
ism by  which  an  organ  or  a  tissue  accommodates  var>-ing  volumes  of  blood.     It  is 
interesting  to  note  that  in  capillary  systems,  notably  that  into  which  the  efferent 
.artery  from  the  glomerulus  breaks  up,  the  capillary  strands  may  vary  in  calibre, 
*  which  would  lead  one  to  expect  that  a  pressure  just  sufficient  to  force  blood  through 


646  DIABETES 

excreted  in  diabetes  is  not  always  proportional  to  the  concentration  of  sugar  in  the 
blood,  but  is  more  nearly  proportional  to  the  product  obtained  by  multiplying 
the  blood  sugar  percentage  by  a  number  representing  the  approximate  blood  volume 
and  other  experiments  to  be  described  later  point  in  the  same  direction. 

The  Utilization  of  Sugar  may  be  considered  for  present  purposes  as 
the  sum  of  the  processes  by  which  a  sugar  such  as  glucose  is  converted 
into  something  else  within  the  cells.  In  the  case  of  glucose  it  includes 
oxidation  to  yield  finally  CO2  and  water;  polymerizatio7i  to  yield  a 
series  of  substances,  chief  of  which  is  glycogen;  reduction  to  fat; 
transformation,  as  to  lactic  acid;  combination,  etc.  The  rate  of  utili- 
zation^ by  all  of  these  methods  taken  collectively  is  influenced  in  the 
first  place  by  the  rate  at  which  glucose  molecules  enter  the  cells  and 
secondly  by  the  reaction  conditions  encountered  within  it.  The 
utilization  rises  with  an  increasing  supply  of  glucose.  As  to  the  factors 
which  enter  into  what  we  have  called  the  reaction  conditions  found 
within  the  cell  there  is  little  definite  knowledge.  With  a  constant 
glucose  supply  the  rate  of  utilization  may  fall  as  the  result  of  a  defi- 
ciency of  that  hypothetical  substance  derived  from  the  pancreas. 
It  is  well  known  that  acid  may  retard  glycogen  formation  and  hasten 
glycogen  hydrolysis.  It  would  appear  from  the  work  of  Murlin  and 
Kramer'^  that  alkali  may  increase  glucose  utilization.  The  rate  of 
actual  oxidation  is  influenced  by  the  supply  of  oxygen,  etc.  The  fol- 
lowing may  serve  to  suggest  other  factors. 

It  might  be  conceived  that  the  cell  contained  molecules  of  a  glucolytic  catalyst 
or  enzyme  similar  in  its  effects  to  metallic  hydroxides,  that  glucose  molecules  as 
fast  as  they  entered  the  cells  would  come  into  collision  with  catalyst  molecules, 
perhaps  combining  with  them,  and  that  as  a  result  of  the  encounter  the  glucose 
molecules  would  be  dissociated  into  unsaturated  fragments  or  ions.  From  the  mo- 
ment of  union  or  dissociation  they  would  cease  to  behave  as  glucose  molecules. 
The  unsaturated  fragments  might  subsequently  suffer  various  fates,  depending 
upon  the  character  and  quantities  of  various  substances  in  the  cell.  Thus,  some 
might  combine  with  oxj^gen  to  yield,  finally,  carbon  dioxide  and  water.  Others 
might  combine  with  each  other  to  form  polymers  like  glycogen,  others  again 
undergo  reduction  to  fat  or  molecular  rearrangement  to  give  lactic  acid.  The 
relative  quantities  undergoing  those  several  changes,  would  depend  upon  the 

the  larger  tubes  would  not  suffice  to  inject  the  smaller,  while  a  rising  pressure  should 
throw  into  action  erstwhile  empty  collaterals  and  vice  versa.  A  system  of  collateral 
spillways  would  limit  the  distension  of  already  filled  capillaries  and  make  the  ac- 
commodations of  varying  blood  volume  largely  a  matter  of  throwing  in  and  cutting 
out  capillary  cylinders.  Such  a  method  if  followed  exclusively  woukl  make  the 
surface  rise  faster  than  the  volume,  since  the  new  clianncls  would  be  of  somewhat 
smaller  diameter.  On  the  other  hand,  if  a  cai)illary  wore  cylindrical,  increasing 
its  volume  by  increasing  its  diameter  would  lessen  the  ratio  of  surface  to  volume. 
Increasing  the  volume  of  a  cylinder  by  increasing  its  length  would  increase  lateral 
surface  in  proportion  to  volinne.  Possibly  in  liealth,  and  within  certain  limits, 
variation  of  the  blood  volume  may  cause  proportional  changes  of  capillarv  surface. 

'  Jour.  Biol.  Chem.,  1914  (18),  21;  Proc.  Soc.  Kxp.  Biol.,  1910  (13)"  07;  also 
"Studies  in  Hyperglycemia  in  Relation  to  Glycosuria,"  Albert  A.  Epstein,  N.  Y,, 
1916. 

"  It  might  not  seem  desirable  to  include  such  processes  as  temporary  storage 
in  the  form  of  glycogen  under  the  heading  of  utilization.  Tlie  term  is  used  for 
convenience. 

'  Jour.  Biol.  Chem.,  1910  (27),  499. 


THE  UTILIZATION  OF  SUGAR  Ml 

relative  quantities  of  II  ami  OH  ions,  of  available  oxyKen,  salts,  etc..  found  in  the 
various  phases  of  the  cell.  This  conception  is  based  on  that  used  by  Nef  to  ex- 
plain the  behavior  of  sugars  in  alkaline  solutions.  For  a  concrete  conception  of 
the  dynamics  of  a  reaction  between  an  organic  substrate  and  catalyst  the  reader 
is  referred  to  Van  Slyke's  study  of  the  enzyme  urea.sc.' 

The  general  principles  outlined  above  may  he  illustrated  by  experi- 
ments with  timed  intravenous  injections  of  glucose.  It  has  long  been 
kno\vn  that  if  a  comparatively  large  dose  of  glucose  is  injected  rapidly 
into  a  peripheral  vein  a  marked  glycosuria  usually  results.  Pavy, 
however,  emphasized  the  fact  that  a  material  fraction  of  a  dose  so 
given  fails  to  be  excreted  and  appears  to  be  utilized.  Doyon  and 
Du  Fourt  demonstrated  that  with  a  standard  dose  of  glucose  the  i)er- 
centages  excreted  and  utihzed  respectively  are  influenced  by  the  time 
consumed  in  injection,  the  slower  rates  of  injection  causing  lower  per- 
centage excretions  and  vice  versa.  Blumenthal  chose  a  standard  in- 
jection time  of  about  10  seconds  and  varied  the  weight  of  sugar  given 
in  that  time.  He  found  that  a  certain  dose  of  glucose  might  be 
injected  into  the  ear  vein  of  a  rabbit  without  causing  any  glycosuria 
at  all.  However,  the  maximum  dose  which  could  be  so  injected  once 
could  not  be  repeated  15  minutes  later  without  causing  glycosuria. 
He  assumed  from  this  that  the  first  dose  "saturated"  the  tissues  and 
that  fifteen  minutes  later  the  utilization  of  sugar  had  only  resulted  in 
a  partial  desaturation.  He,  therefore,  determined  the  dose  of  glucose 
which  might  be  injected  repeatedly  at  15  minute  intervals  for  as  long 
as  3  hours  without  ever  causing  glycosuria.  His  figures  varied  be- 
tween 0.6  and  1.3  gm.  per  kg.  of  body  weight  per  hour.  This  he 
termed  the  "utilization  limit,"  whereas  the  largest  dose  which  could 
be  given  within  10  seconds  once  without  causing  glycosuria  he  called 
the  "saturation  limit."  The  latter  he  placed  at  0.8  gm.  per  kg.  but 
R.  M.  Wilder  has  been  unable  to  confirm  this  latter  observation. 
Woodyatt,  Sansum  and  Wilder^  made  continued  intravenous  injections 
of  glucose  at  uniform  rates  by  means  of  a  motor  driven  pump  for  2  to 
17  hours  with  the  following  findings: 

If  chemically  pure  glucose  in  aqueous  solution  is  injected  con- 
tinuously into  the  peripheral  venous  blood  of  a  normal  resting  man, 
dog  or  rabbit  at  the  rate  of  0.8  gm.  per  kg.  of  body  weight  per  hour, 
or  at  any  slower  rate,  the  injection  may  be  sustained  in  most  cases, 
hour  after  hour  for  7  hours  and  probably  longer  without  producing 
any  glycosuria  in  the  usual  sense  of  the  word.  If  the  rate  is  advanced 
to  0.9  gm.  of  glucose  per  kg.  of  body  weight  per  hour,  while  all  other 
conditions  remain  the  same,  the  injection  may  be  sustained  for  a  short 
time  without  causing  glycosuria,  but  in  nearly  all  cases  abnormal 
quantities  of  glucose  begin  to  appear  in  the  urine  after  5  to  30  minutes 

8  Jour.  Biol.  Chem.,  1914  (19),  141. 

9  Jour.  Amer.  Med.  Assoc,  1915  (G5),  2067  (preliminary  report);  \\oodyatt, 
Harvey  Society  Lectures,  191G;  Wilder  and  Sansum,  Arch.  Int.  Med.,  1917  (19), 
311;  Woodyatt  and  Sansum,  Jour.  Biol.  Chem.,  1917  (;30),  155. 


648  DIABETES 

of  injection.  Once  established,  the  glycosuria  then  tends  to  proceed  at 
a  uniform  rate  as  long  as  the  rate  of  injection  and  other  conditions  re- 
main fixed.  However,  if  the  injection  rate  is  again  reduced  to  0.8  gm. 
per  kg.,  glycosuria  promptl}^  ceases.  Thus  during  the  continuance  of 
an  injection  at  the  latter  (0.8  gm.)  rate  there  can  be  no  continued 
accumulation  of  unchanged  glucose  in  the  body,  but  the  rate  of  injec- 
tion is  equalled  by  the  rate  of  utilization  if  we  leave  out  of  considera- 
tion the  trace  of  sugar  which  can  be  detected  in  the  urine  by  refined 
methods.  It  is  important  to  note  that  it  makes  no  appreciable  differ- 
ence whether  one  uses  an  18  or  a  72  per  cent,  glucose  solution  for  in- 
jection. The  tolerance  limit  for  glucose  may  be  demonstrated  at  the 
same  point  regardless  of  wide  variation  in  the  quantity  of  water  ad- 
ministered with  the  glucose,  even  though  the  blood  volume  and  the 
blood  sugar  percentages  may  be  influenced  by  variation  of  the  water 
supply.  Also,  if  glucose  is  injected  continuously  and  uniformly  at  a 
rate  productive  of  some  glycosuria,  the  glucose  excretion  may  proceed 
at  a  constant  rate  in  spite  of  marked  variation  in  the  water  supply 
during  successive  hours.  A  certain  dog  receiving  by  vein  20  gm.  of 
glucose  per  10  kg.  per  hour  for  8  hours,  excreted  every  hour  close  to 
0.42  gm.  of  sugar  per  10  kg.  of  body  weight.  Yet  during  the  ex- 
periment water  was  injected  at  varying  rates  into  the  same  vein  with 
the  glucose,  so  that  the  hourly  volume  of  urine  varied  between  6  c.c. 
and  128  c.c.  and  the  percentages  of  sugar  in  the  urine  varied  between 
0.35  and  4.9.  This  emphasizes  the  fundamental  importance  of  the 
rate  at  which  sugar  is  supplied  to  the  organism  in  determining  the 
occurrence  or  non-occurrence  of  glycosuria  and  in  fixing  the  rate  of 
excretion  when  the  latter  occurs. 

In  view  of  the  above  generalities  several  specific  mechanisms  sug- 
gest themselves  by  which  glycosuria  might  be  produced : 

(1)  An  increased  supply  of  preformed  glucose  to  the  whole  organ- 
ism from  without  (alimentary  glycosuria). 

(2)  A  decreased  utilization  in  the  organism  as  a  whole  (pancreatic 
diabetes) . 

(3)  An  increased  supply  to  the  kidneys  resulting  from  the  hbera- 
tion  into  the  blood  of  sugar  previously  stored  or  combined  in  other 
organs.  Thus,  the  rapid  hydrolysis  of  glycogen  following  puncture 
of  the  floor  of  the  fourth  ventricle  and  analogous  nerve  stimulations, 
and  occurring  in  the  acid,  asphyxial,  narcotic,  thja-oid,  epinephrine, 
and  hypophysis  glycosurias.  In  an  analogous  manner  lactose  may 
enter  the  circulating  blood  from  the  mammary  gland,  and  pentose 
from  unknown  sources. 

(4)  An  increased  supply  to  the  kidneys  due  to  decreased  utilization 
in  other  organs.  The  breaking  down  of  glycogen  mentioned  in  (3) 
might  be  so  interpreted  and  one  might  think  of  the  possibility  that  in 
various  diseases  the  ability  of  a  part  to  utilize  sugar  maj'^  be  altered. 

(5)  Decreased  utilization  in  tlu^  kidney  itself. 


THE  BLOOD  SUGAR  649 

(6)  Increased  physical  pciictnibility  of  tlie  kidney  membrane  to 
glucose. 

Both  (5)  and  (6)  arc  hypothetical  conditions,  the  latter  having 
been  proposed  as  the  basis  of  so  called  kidney  diabetes,  a  state  in 
which  glycosuria  occurs  with  a  normal  or  subnormal  percentage  of 
sugar  in  the  blood,  in  which  the  rate  of  sugar  excretion  is,  in  com- 
parison with  other  forms  of  glycosuria,  little  influenced  by  the  diet,'" 
and  in  which  the  glycosuria  tends  to  grow  progressive!}'  worse. 

THE  BLOOD  SUGAR 

The  normal  blood  sugar  concentration  is  found  to  average  0.10 
per  cent.,  but,  as  statistics  show,  it  may  vary  at  least  between  0.06  and 
0.11  per  cent.  The  literature  contains  numerous  references  to  that 
blood  sugar  concentration  which  if  exceeded  leads  to  glycosuria 
("threshhold"  value).  In  accordance  with  the  general  principles 
above  discussed  we  should  expect  this  value  to  vary.  It  has  been 
placed  at  0.147  to  0.164  per  cent,  by  Foster,  between  0.17  and  0.18 
per  cent,  by  Haman  and  Hirschman,  at  about  0.20  per  cent,  by  Pavy, 
and  other  writers  have  reported  greater  variations,  due  in  part  doubt- 
less to  differences  in  the  analytical  methods  used.  How  widelj'  the 
threshhold  blood  sugar  percentage  may  be  varied  by  extreme  variations 
of  the  blood  volume  and  other  factors  has  not  been  settled.  Following 
the  ingestion  of  free  glucose  the  blood  sugar  percentage  ordinarily 
rises,  and  in  a  similar  way,  but  more  slowly,  after  feeding  of  starch. 
Fisher  and  Wishart  gave  50  gm.  of  glucose  in  150  c.c.  of  water  by 
stomach  to  dogs  weighing  8  to  9  kg.  and  found  in  the  first  hour  blood 
sugar  percentages  of  0.16  and  0.13.  In  succeeding  hours  there  was 
little  variation  from  0.11  per  cent.  In  harmonj^  with  the  previous 
work  of  Gilbert  and  Baudoin  and  the  more  recent  studies  of  others  on 
man,  these  experiments  showed  that  the  blood  sugar  percentage  rises 
during  the  first  hour,  then  falls  and  thereafter  remains  normal.  There 
was  no  increase  of  the  blood  volume  during  the  first  hour,  the  hemo- 
globin percentage  remaining  unchanged,  probably  because  the  large 
quantity  of  glucose  in  the  bowel  held  water  there.  But  in  the  second 
hour  the  blood  volume  became  large  and  the  hemoglobin  showed  the 
effects  of  dilution.  In  this  same  hour  the  sugar  percentage  returned 
to  normal.  But  the  absorption  of  glucose  was  only  completed  in  the 
fourth  hour  and  calorimetric  observations  by  Lusk  showed  that  the 
metabolism  also  ran  at  a  uniform*  rate  20  per  cent,  above  the  basal 
level  into  the  fourth  hour.  Accordingly  the  observed  blood  sugar  per- 
centages first  rose  as  the  rate  of  sugar  supply  was  increased,  but  fell 
again  during  the  maintenance  of  the  increased  supply  and  while  the 
metabolism  was  constant,  owing  to  the  shifting  of  water. 

When  concentrated  (54  to  72  per  cent.)  glucose  solutions  are  in- 

5"  Cf.  Epstein;^  Strouse,  Jour.  Amer.  Med.  Assoc.  1914  (G2),  1301;  Lewis  and 
Mosenthal,  Johns  Hopkins  Hosp.  Bull.,  191G  (27),  133. 


650  DIABETES 

jected  continuously  into  the  blood  at  rates  of  0.4  to  0.8  gm.  per  kg. 
per  hour,  there  is  at  first  a  steep  rise  of  the  blood  sugar  percentage, 
followed  by  a  fall  coincident  with  an  increased  hydremia,  after  which 
a  new  equilibrium  is  established  and  the  blood  sugar  percentage  may 
become  constant  at  a  "normal"  level  exactly  as  in  the  above.  By 
injecting  glucose  at  the  same  rates  in  sufficiently  dilute  solutions  this 
initial  rise  may  be  very  much  reduced  and  the  blood  sugar  percentage 
established  in  later  hours  may  even  be  lower  than  that  observed  before 
injection  began.  On  the  other  hand,  if  glucose  is  injected  at  rates 
above  0.9  gm.  per  kg.  per  hour,  glycosuria  begins,  and  if  the  rate  of 
injection  is  rapid  enough  may  be  made  intense.  As  glucose  passes 
through  the  kidney  membrane,  water  tends  to  accumulate  with  the 
glucose  on  the  urinary  side  of  the  membrane  (increased  diuresis, 
polyuria) .  In  the  same  way  that  glucose  in  the  bowel  lumen  may  tend 
to  withhold  water  from  the  blood,  so  a  sufficient  quantity  of  glucose 
in  the  urinary  tubules  may  manifest  the  same  tendency  in  this  local- 
ity. Whether  the  glucose  in  the  urinary  tubules  will  have  the  effect 
of  concentrating  the  blood  or  vice  versa  will  depend  on  the  quantitative 
distribution  of  free  sugar  between  these  two  fluids,  and  the  quantity 
of  water  available  for  distribution  between  the  blood  sugar  and  the 
urinary  sugar.  During  continuous  intravenous  injections  of  glucose 
at  rates  from  2.7  gm.  per  kg.  per  hour  upward,  30  to  40  per  cent, 
of  the  glucose  injected  may  be  excreted  and  there  is  a  strong  tendency 
toward  dehydration  of  the  whole  body.  This  may  be  neutralized  by 
supplying  water  with  the  sugar  as  fast  as  it  flows  away  in  the  urine, 
provided  the  rate  of  injection  is  not  so  great  that  the  necessarj^  traffic 
in  water  overtaxes  the  cardio-renal  mechanism.  By  employing  these 
high  rates  of  injections  and  maintaining  the  water  balance  at  as  low 
levels  as  compatible  with  life  and  recovery,  it  is  possible  to  produce 
and  maintain  for  hours  blood  sugar  concentrations  as  high  as  2.38  per 
cent.  Joslin  observed  1.49  per  cent,  of  sugar  in  the  blood  of  a  fatal 
case  of  diabetes  with  nephritis.  The  blood  sugar  of  diabetics  passing 
sugar  in  the  urine  is  as  a  rule  higher  than  normal,  but  not  necessarily 
so,  much  depending  on  the  water  balance.  Joslin's  statistics  show  a 
range  of  0.07  to  0.43  per  cent. 

THE  STATE  OF  THE  SUGAR  IN  THE  BLOOD 

It  has  long  been  .believed  that  the  sugar  circulating  in  the  blood 
exists  in  two  physical  states,  a  diffusible  and  a  non-diffusible,  i.  e., 
as  (a)  Free  glucose  in  a  state  comparable  to  that  of  glucose  dissolved  in 
water,  sucr6  actuelle  of  Lupine,  (b)  Sugar  in  a  colloid  state,  sucre 
virtuelle,  Lupine.  By  the  former  term  a  very  specific  idea  is  conveyed. 
One  might  think  for  instance  of  single  molecules  or  clusters  of  two  or 
three,  each  holding  in  its  sphere  of  influence  a  certain  number  of  water 
molecules  like  suns  in  solar  sj'stems.  Sucli  sniall  masses  move  at 
high  velocities,  "diffuse"  readily  and  create  high  "osmotic  pressures." 


I 


THE  liLOOl)  SrCAU  (;.')! 

B}'  the  latter  term  is  meant  sugar  in  the  blooti  wliich  does  not  lj(;liavo 
physically  like  glucose  in  aqueous  solution  nor  respond  to  the  ordinary 
chemical  tests  for  sugar,  but  from  which  free  glucoses  may  be  reliberated 
by  such  simple  procetlures  as  boiling  witli  dilute  a(;ids.  Such  sugar 
is  supposed  to  exist  as  a  eomponcMit  of  particles  having  tlu;  larger  di- 
mensions which  characterize  colloids  (non-difTusoids).  But  as  to 
the  actual  chemical  nature  of  these  a  great  variety  of  proposals  have 
been  made.  Thus  Pavy  proposed  glucose  molecules  held  entire  to 
the  colloids  of  the  blood  in  a  state  of  simple  adsori)tion  (com[)arable 
to  the  state  of  molecules  of  a  dye  electrically  bound  to  particles  of  a 
colloid  clearing  agent).  He  also  proposed  glucose  chemically  incor- 
porated in  the  structure  of  the  protein  molecule,  and  between  these 
extremes  by  the  same  author  a  score  of  suppositions  have  l)een  made  by 
others,  among  which  Drechsel's  jecorin,  a  lecithin-sugar  comi)ound, 
is  a  notable  example.  Another  worthy  of  serious  consideration  is 
that  of  glucose  built  up  into  polymers  intermediate  between  disac- 
charides  and  glycogen.  The  ba?is  for  assuming  the  existence  of  com- 
bined sugar  in  the  blood  lies  chiefly  in  the  observation  that  following 
glucose  administrations  the  increase  in  the  reducing  power  of  the  blood 
which  results  from  heating  the  blood  with  dilute  acid  is  greater  than 
the  increase  resulting  from  the  same  process  before  sugar  admin- 
istration (see  Pavy,  Lepine,  Loewi).  Also,  if  glucose  is  added  to  fresh 
blood  and  the  mixture  placed  in  the  incubator,  the  reducing  power 
falls  but  may  be  in  part  rehabihtated  by  boihng  with  dilute  acid.^" 
As  to  the  sugar  which  is  determined  by  the  ordinary  methods  of 
blood  analysis,  it  would  appear  that  we  are  dealing  almost  exclusively 
with  free  glucose.  As  yet  no  one  has  succeeded  in  proving  the  exist- 
ence in  blood  of  a  combined  sugar  capable  of  spontaneous  conversion 
into  free  sugar.  Michaelis  and  Rona  dialyzed  separate  portions  of  the 
same  blood  against  isotonic  salt  solutions  containing  graduated  quan- 
tities of  sugar.  A  sugar  solution  which  neither  lost  nor  gained  sugar 
during  the  dialysis  they  regarded  as  having  an  amount  of  free  sugar 
equal  to  that  in  the  blood.  Titration  of  the  blood  sugar  and  of  the 
sugar  in  such  a  solution  gave  almost  identical  figures.  They  accord- 
ingly concluded  that  all  of  the  reducing  sugar  in  this  blood  muse  have 
been  as  free  to  diffuse  as  was  that  in  the  simple  salt  solution.  But  this 
ingenious  experiment  of  Michaelis  and  Rona  does  not  show  conclusively 
that  in  circulating  blood  there  is  no  sugar  in  a  state  of  colloidal 
adsorption,  because  drawn  blood  rapidly  undergoes  survival  changes 
(e.  g.,  lactic  acid  formation)  which  might  influence  the  affinity  of  its 
colloids  for  sugar.  However,  McGuigan  and  Hess"  led  the  blood  of 
living  animals  through  collodion  tubes  enclosed  in  jackets  filled  with 
isotonic  salt  solutions  and  found  that  when  equiUbrium  was  established 

1"  A  critical  review  of  the  literature  to  1912  will  be  found  in  the  books  by 
McLeod  and  Allen.  Compare  also  the  article  by  Levene  and  the  recent  studies 
of  Lombroso  favoring  the  polvnierization  idea. 

"  Jour.  Pharm.  and  Exp.  Ther..  (1914)  (ti),  45. 


652  *  DIABETES 

the  concentration  of  reducing  sugar  in  the  salt  solution  and  in  the 
plasma  was  the  same,  proving  that  even  in  life  all  of  the  titrable  plasma 
sugar  is  in  a  state  of  subdivision  which  lets  it  migrate  through  the  inter- 
stices of  a  collodion  membrane.  This  would  make  the  adsorption 
idea  seem  untenable. 

Closely  related  to  the  question  of  the  state  of  the  sugar  in  the  blood 
is  that  of  its  state  in  the  cells.  Palmer^^  has  studied  the  percentages 
of  sugar  found  in  the  various  tissues  in  relationship  to  the  plasma 
sugar  concentration.  The  titrable  sugar  of  the  tissues  was  found  below 
that  of  the  blood  in  all  organs  except  the  liver.  Of  course,  owing 
to  the  large  quantities  of  glycogen  which  occur  in  that  organ  and  the 
rapidity  with  which  it  breaks  down  into  glucose,  liver  tissue  would 
naturally  analyze  high  for  sugar.  In  the  muscles  the  titrable  sugar 
was  found  at  0.04  and  0.041  per  cent,  with  blood  sugar  at  0.10  and 
0.105  per  cent.  On  the  other  hand  the  tissues  generally  when  boiled 
with  dilute  acid  show  a  higher  content  of  "combined"  sugar  than  the 
blood.  This  is  most  striking  in  the  case  of  the  liver  and  due  by  com- 
mon consent  to  the  polymers  of  glucose  in  that  organ. 

It  serves  a  useful  purpose  to  consider  the  body  as  a  whole  as  an 
heterogeneous  system  made  up  of  phases,  and  to  assume  that  glucose 
on  entering  the  body  distributes  itself  between  these  phases  as  acetic 
acid  may  distribute  itself  between  the  fat  droplets  and  the  aqueous 
part  of  milk;  that  glucose  in  a  certain  type  of  phase  behaves  as  though 
in  water  and  in  another  type  of  phase  rapidly  undergoes  chemical 
changes.  The  blood  is  a  tissue  in  which  the  dominant  phase  is  in  the 
nature  of  a  physical  solvent  for  glucose,  like  water.  In  the  cells  the 
dominant  phases  are  of  such  a  character  that  glucose  on  entering  them 
rapidly  undergoes  chemical  change.  But  both  types  of  phase  are 
present  in  both  blood  and  cells  although  in  different  proportions. 
The  blood  is  therefore  the  phase  par  excellence  in  which  to  study  the 
"  Sucre  actuelle"  and  the  tissues  the  place  to  study  the  "sucr6  vir- 
tuelle."  According  to  this  conception  "sucr4  virtuelle"  would  be 
glucose  in  process  of  utilization  or  storage  and  not  beyond  recall, 
hence  chiefly  glucose  polymers. 

DIOSE'3 

Diose,  glycollic  aldehyde^  CH2OH-COH,  the  simplest  sugar,  of  which  there  is 
but  one  possible  form,  is  highly  sensitive  to  oxidative  influences  and,  in  vitro, 
readily  condenses  with  alkali  to  yield  a  complex  mixture  of  higher  sugars  .and 
saccharinic  acids  in  a  manner  analogous  to  that  manifested  by  the  trioses.  Not- 
withstanding its  instability  and  sensitiveness  to  oxidative  changes  in  the  tost 
tube,  it  would  appear  that  glycollic  aldehyde  is  insusceptible  of  direct  oxidation 
in  the  body  but  that  it  may  be  converted  into  glucose,  like  other  sugars,  and  then 
utilized.     When  given  intravenously  at  the  rate  of  only  0.1  gm.  per  kg.  per  hour, 

"Jour.  Biol.  Chem.,  1917  (30),  79. 

"Literature  on  diose-  Mayer,  P.,  Zeit.  f.  phAsiol.  Chem.,  1903  (3S\  135; 
Woodyatt,  R.  T.,  Jour.  Amer.  Med.  Assoc,  1910  (r).!),  2109;  Tarnas  and  Haer, 
Biochein.  Zeit.,  1912  (41),  38G;  Smedley,  Ida,  Jour.  Thysiol.,  1912  (44),  203; 
Sansum,  W.  D.  and  Woodyatt,  R.  T.,  Jour.  Biol.  Chem.,  1914  (17),  521. 


DIOSES  AX  I)  TRIOSES  653 

unchanged  diose  appears  in  the  urine  after  the  first  few  minutes  of  injection 
(author).  P.  Mayer  reported  glycosuria  and  death  following  administration  of 
impure  glycollic  aldehyde  to  rabbits.  Parnas  and  Haer  saw  an  increase  of  glycogen 
in  tortoise  livers  perfused  with  glycollic  aldehyde.  This  is  confircd  by  Harren- 
scheen.  Smedley  noted  the  rapid  disappear;ince  of  diose  added  to  liver  emulsions. 
Sansum  and  Woodyatt,  and  also  Greenwald  obscr\-ed  slight  increases  of  the 
glycosuria  following  parenteral  administrations  of  diose  in  phlorhizinized  but  not 
completely  dcglycogenized  dogs.  The  e.xtra  sugar  could  have  come  from  glycogen 
in  these  experiments.  A  final  proof  of  the  conversion  of  diose  into  glucose  in  the 
living  body  has  not  been  brought.  The  relationship  of  this  substance  to  glycine, 
CH2NH2-COOH;  glycoUic  acid,  CH2OH-COOH;  and  ethyl  alcohol,  CH,-CH,OH; 
is  close.  Lusk  states  that  glycine  is  capable  of  conversion  into  glucose  in  the  body. 
However,  glycollic  acid  and  alcohol  are  apparently  not  sugar  formers. 

TRIOSES'* 

There  are  three  possible  trioses,  d-  and  1-glyceric  aldehyde  and  the  ketotriose 
dihydroxyacetone.  The  optically  inactive  d,  1-glyceric  aldehyde  has  been  prepared 
and  recently  the  d-  and  l-forms.  The  preparation  is  still  tedious  and  expensive. 
Dihydroxy-acetone  is  somewhat  easier  to  prepare.  Both  trioses  are  unstable, 
easily  oxidized  and  very  prone  to  undergo  rearrangements  and  condensation  with 
even  traces  of  alkali.  Under  the  influence  of  alkali  they  yield  complex  mixtures  of 
hexoses,  chiefly  3-ketohexoses,  formerly  known  as  a  and  ^-acrose  from  which 
Schmitzi*  has  recently  isolated  d,l-fructose  and  d,l-sorbose.  If  o.xygen  is  avail- 
able as  well  as  alkali,  they  burn.  If  the  alkali  is  strong  and  oxygen  lacking,  much 
lactic  acid  is  formed  together  with  certain  rearranged  tetrose,  pentose  and  hexose 
molecules,  known  as  saccharinic  acids  (or  "saccharines,"  of  Iviliani).  The  same 
phenomena  occur  when  the  alkali  is  dilute,  but  more  slowlj'.  The  structural 
formulae  of  the  trioses  and  their  relationship  to  glycerol,  glyceric  acid  and  lactic 
acids  (the  latter  of  which  might  be  regarded  as  a  3-carbon  saccharinic  acid)  may 
be  seen  from  the  following  chart: 

H  ]        H  H  H  OH  OH  OH 

'  I  ^  I  I 

H— C— OH         C=0  C=0  H— C— OH         C^O  C=0  C^O 

I  I  i  I 

H— C— OHH— C— OHHO— C— H  C     O  H— C— OH  H— C— OH  HO— C— H 

j 

H— C— OH  H— C— OH     H— C— OH  H— C— OH  H— C— OH  H— C— H       H— C— H 

II  •  . 

H  H  H  H  H 

1-glyceric  dihydrosy  d-glyceric       d-lactic  acid.     I-lactic  acid. 

aldehyde  acetone  acid 


H 

H 

Glycerol 
(alcohol) 

d-glyceric 
aldehyde 
(aldose) 

(aldose)  (ketose)  3-carbon  saccharinic 

acid 

Neuberg  fed  animals  and  men  considerable  doses  of  impure  d.l- 
glyceric  aldehyde  (glycerose)  and  saw  its  apparently  complete  util- 
ization. Parnas  demonstrated  increased  glycogen  in  tortoise  livers 
perfused  with  d,l-glyceric  aldehyde.  Smedley  noted  the  rapid  chs- 
appearance  of  glyceric  aldehyde  added  to  liver  emulsions.  Sansum 
and  Woodyatt  fed  pure  crystalline  d,l-glyceric  aldehyde  to  rabbits 
and  guinea  pigs  in  doses  as  high  as  2.8  gm.  per  kg.  with  no  apparent 
ill  effects.     A  dose  of  5  gm.  per  kg.  in  a  rabbit  caused  diarrhea  with 

"  Literature  on  Trioses:  The  chemical  literature  is  reviewed  and  an  improved 
method  of  preparing  glvceric  aldehyde  described  by  Witzemann,  E.  J.,  Jour.  Am. 
Chem.  Soc,  1914  (36)",  1908,  and  ibid.,  p.  2223.  The  biological  literature  is 
reviewed  by  Sansum,  W.  D.  and  Woodyatt,  R.  T.,  Jour.  Biol.  Chem..  1916  (24), 
327. 

i^Ber.  Deut.  Chem.  Ges.,  1914  (46),  2327. 


654  DIABETES 

unchanged  triose  in  the  passages.  There  was  marked  diminution  of 
urine  with  albuminuria,  which  then  persisted  for  10  days.  A  dose 
of  6.8  gm.  per  kg.  killed  in  4  hours.  In  no  case  was  there  an  alimentary 
triosuria.  The  average  lethal  dose  by  the  subcutaneous  route  was 
2.2  gm.  per  kg.  as  compared  with  18  gm.  of  glucose  per  kg.  in  the 
same  set  of  animals.  Suppression  of  urine  is  a  regular  manifestation, 
but  the  visceral  changes  at  autopsy  are  slight.  When  d,l-glyceric 
aldehyde  is  injected  intravenously  at  the  rate  of  only  0.15  gm.  per  kg. 
per  hour,  and  possibly  at  slower  rates,  unchanged  glyceric  aldehyde 
appears  in  the  urine,  but  no  glucose.  (It  will  be  recalled  that  glucose 
may  be  injected  continuously  at  the  rate  of  0.8  gm.  to  0.9  gm.  per  kg. 
per  hour  without  causing  glycosuria.)  When  administered  to  diabetic 
individuals  d,l-glyceric  aldehyde  may  increase  glycosuria.  When 
given  to  completely  phlorhizinized  and  glycogen-free  dogs  it  is  pos- 
sible to  demonstrate  a  quantitative  conversion  of  the  triose  into  glucose, 
the  increase  in  glycosuria  corresponding  exactly  with  the  weight  of 
glyceric  aldehyde  given.  However,  owing  to  the  toxic  effects  of  gly- 
ceric aldehyde  on  the  kidneys  there  may  be  an  incomplete  excretion 
of  all  the  sugar  formed.  The  suppression  of  urine  has  in  the  past 
been  mistaken  for  a  beneficial  effect,  since  it  may  lead  to  diminished 
excretions  of  sugar,  acetone,  aceto-acetic  and  /3-hydroxybutyric  acids. 

Embden  and  his  coworkers  demonstrated  the  formation  of  lactic 
acid  from  glyceric  aldehyde  added  to  washed  blood  corpuscles.  The 
keto-triose,  dihydroxyacetone,  was  observed  to  produce  less  lactic  acid, 
but  otherwise  it  is  not  improbable  that  the  behavior  of  the  ketotriose 
is  analogous  to  that  of  the  aldo  forms.  Thus  Mostowski  found  dihy- 
droxyacetone to  be  a  glycogen  former,  and  Ringer ^^  reported  its  com- 
plete  transformation   into   glucose   in  the  fully  phlorhizinized  dog. 

The  complete  conversion  of  d,l-glyceric  aldehyde  into  glucose  in 
phlorhizinized  dogs — its  transformation  into  glycogen  in  the  perfused 
liver,  its  disappearance  as  such  when  added  to  liver  emulsions,  all 
indicate  that  glyceric  aldehyde  (like  diose  and  other  sugars  in  general) 
is  converted  into  glucose  in  the  body  as  a  preliminary  stej)  in  utili- 
zation. The  fact  that  large  doses  may  be  given  by  the  alimentary 
route  without  causing  melituria  or  death,  whereas  much  snuiller  doses 
given  subcutaneously  may  prove  lethal,  together  with  the  verj'  low 
rate  at  which  glyceric  aldehyde  has  to  be  given  by  vein  in  order  not  to 
produce  melituria,  all  point  to  the  liver  (and  bowel  wall)  as  the  chief 
sites  of  its  conversion.  Glyceric  aldehyde  has  figured  prominently 
in  theories  of  the  normal  catabolism  of  glucose,  antl  on  the  basis  of  his 
observations  concerning  the  formation  of  lactic  acid  from  this  triose 
by  blood  corpuscles  Embden  regards  it  as  a  chief  normal  intermediate 
substance  in  the  oxidation  of  glucose  in  the  cells.  Now  glucose  may 
be  oxidized  in  the  body  at  the  rate  of  0.0  gm.  per  kg.  per  hour  under 
suitable  circumstances,  and  if  every  molecule  of  glucose  oxidized  were 

»  Ringer  and  Frankel,  Jour.  Biol.  Cheni.,  1914  (IS),  233. 


TRIOSES  0.')5 

first  split  to  ffive  two  molecules  of  glyceric  aldehyde,  as  the  iunlnlen 
hypothesis  would  demand,  then  glyceric  aldehyde  would  be  formed 
in  the  body  at  the  rate  of  O.G  gm.  per  kg.  per  hour,  and  tlu;  i)lace  of 
formation  would  be  within  the  cells  of  the  body  at  large,  the  muscles 
representing  the  most  important  sites  of  oxidation.  However,  if  gly- 
ceric aldehyde  is  introduced  into  the  systemic  blood  at  only  one-fourth 
of  this  rate,  unchanged  triose  appears  in  the  urine  and  may  be  demon- 
strated in  the  blood.  But  gljxeric  aldehyde  has  never  been  foimd  in 
the  blood,  urine  or  tissues  under  any  other  circumstances.  Cllyceric 
aldehyde  may  of  course  enter  the  body  via  the  portal  route  at  faster 
rates  without  causing  triosuria,  but  then,  as  stated,  it  would  appear 
not  to  be  oxidized  directly  but  first  assimilated,  i.  e.,  transformed  into 
glucose.  Recently,  for  other  reasons,  von  Fiirth'^  has  also  questioned 
the  tenability  of  Embden's  h3^pothcsis. 

Lactic  acid  from  triose:  When  alkali  acts  on  glucose  (or  hexoses  in  general) 
in  the  absence  of  oxygen,  lactic  acid  is  formed  in  amounts  as  high  as  40  to  60 
per  cent,  of  the  weight  of  the  sugar  used,  provided  the  conditions  are  properly 
controlled.  But  in  the  presence  of  sufficient  oxygen  no  lactic  acid  is  formed. 
Still,  preformed, lactic  acid  will  not  be  destroyed  if  it  is  added  to  this  latter  mix- 
ture. So  it  is  clear  that  lactic  acid  is  not  an  intermediate  in  the  oxidative  break- 
down of  sugars  in  the  alkaline  solution.  Meisenheimer  accordingly  suggested 
the  obvious  probability  that  there  was  some  labile  precursor  of  lactic  acid  which 
burned  in  the  presence  of  oxygen;  in  the  absence  of  oxygen,  rearranged  to  give 
lactic  acid.  He  proposed  glyceric  aldehyde  as  such  a  body.  Nef,  however,  holds 
that  the  immediate  precursor  of  lactic  acid  is  methyl  glj-oxal  (CH3 — CO — COH), 
which  forms  lactic  acid  by  undergoing  what  is  known  to  chemists  as  a  "benzilic 
acid  rearrangement."  These  phenomena  are  remarkably  similar  to  those  that 
occur  in  the  body. 

One  other  important  point  should  be  emphasized  in  this  place.  The  trioses 
condense  in  the  presence  of  alkali  to  yield  among  other  things  certain  hexoses, 
and,  as  described  under  hexoses — any  sugar  of  that  type  will  in  the  presence  of 
alkali  enter  into  a  complex  equilibrium  with  several  other  hexoses.  Any  of  these 
may  again  split  into  3-carbon  compounds  such  as  the  trioses  and  then  again  con- 
dense, and  so  on,  as  long  as  they  do  not  become  converted  into  lactic  acid  or  the 
saccharinic  acids — substances  which  are  not  reconvertible  into  sugar.  Xef  for- 
mulated the  view  that — were  it  not  for  the  occurrence  of  these  irreversible  reactions 
—any  sugar  in  the  presence  of  alkali  would  come  finally  to  represent  an  equilibrium 
of  every  possible  sugar  of  2  to  6  carbon  atoms  {i.  e.,  56)  together  with  all  of  the 
myriad  intermediate  forms.  In  the  body,  however,  lactic  acid  can  be  converted 
into  sugar.  So  this  bar  to  the  great  equilibrium  is  there  nonexistent,  and  it  is 
conceivable  that  in  the  body  there  actually  exists  an  equilibrium  of  this  sort. 

In  all  of  Embden's  experiments  there  was  perhaps  a  lack  of  oxygen, 
so  that  the  phenomena  in  vivo  and  in  alkaline  solution  in  vitro  are  strik- 
ingly parallel.  Embden,'  on  the  basis  of  these  experiments,  regards 
sarcolactic  acid  (i.  e.  d-lactic)  as  a  c/ize/wormaZ  breakdown  product  of 
glucose  in  the  body  over  the  glyceric  aldehyde  route.  But  it  is  hard 
to  see  why  this  assumption  is  more  rational  than  it  would  be  to  say  that 
lactic  acid  is  an  intermediate  in  the  oxidative  breakdown  of  sugars  in 
the  alkaline  solution  outside  the  body,  which  it  certainly  is  not.  Al- 
though lactic  acid  will  disappear  from  a  surviving  asphyxiated  muscle 
if  oxygen  be  resupphed  to  it  (Fletcher)  1^"  and  although  this  disappear- 

1'  Biochem.  Zeit.,  1916  (69),  199. 
1^"  Jour,  of  Physiol.,  1907  (35),  247. 


656  DIABETES 

ance  will  not  occur  in  a  simple  alkaline  peroxide  solution  it 
may  nevertheless  be  effected  by  the  addition  of  a  second  catalyst, 
and  still  we  know  that  the  lactic  acid  was  not  an  intermediate  in 
the  original  solution  until  the  oxygen  supply  became  deficient.  Lactic 
acid  is  probably  an  intermediate  in  the  sugar  cataboHsm  only  during 
relative  asphyxia. 

All  the  substances  whose  formulae  are  given  above  have  been  shown 
to  be  capable  of  conversion  into  glucose  in  the  body.  The  details 
of  the  steps  involved  have  not  been  established,  but,  in  conformity 
with  the  chemical  theories  developed  by  Nef  and  discussed  under 
hexoses,  the  transformation  of  these  substances  into  glucose,  as  well 
as  their  occurrence  in  the  course  of  its  breakdown,  are  best  explained 
on  the  basis  that  all  of  them  participate  in  the  same  great  chemical 
equilibrium  with  the  sugars,  and  that  this  participation  depends  upon 
their  dissociation  into  unsaturated  residues.  These  residues  are  in 
dynamic  chemical  equilibrium  with  the  molecules  from  which  they 
are  derived  and  with  those  derived  from  sugars.  When  there  is  a 
rapid  loss  of  glucose  from  the  body  these  substances  tend  to  become 
glucose,  in  accordance  with  the  laws  of  chemical  equilibrium. 

TETROSES 

There  are  six  possible  tetroses  (4  aldo-  and  2  keto-).  The  entire  subject  of 
their  physiology,  which  has  undoubtedly  considerable  biologic  importance,  has 
been  little  studied.  They  have  never  been  found  in  the  urine,  since  there  are  at 
present  no  established  methods  for  their  detection,  and  efforts  have  been  lacking. 

PENTOSES 

Chemical  theory  demands  the  existence  of  fourteen  pentoses,  i.  e.,  six  aldo- 
pentoses,  four  2-keto-pentoses  and  four  3-keto-pentoses.  Only  those  better 
known  to  chemists  have  received  biological  study,  e.  g.,  arabinose  and  xylose.^* 
Of  these  the  optically  inactive  or  d-  1-arabinose,  the  1-arabinose  and  1-xylose  are 
the  best  known.  When  even  small  quantities  of  pentose  gain  access  to  the  cir- 
culating blood,  pentose  is  excreted  in  the  urine.  Ebstein^^  reports  the  appearance 
of  traces  in  the  urine  of  a  man  (in  which  none  had  been  previously  demonstrated), 
after  the  administration  of  so  little  as  0.25  gram  of  1-arabinose  by  mouth.  Ber- 
gelP"  found  reactions  for  pentose  in  the  urine  seven  to  ten  minutes  after  ingestion 
of  the  same  sugar,  and  when  given  subcutaneously.  Fr.  Voit^^  saw  about  50  per 
cent,  excreted.  Neuberg  and  Wohlgemuth"  gave  a  normal  man  15  grams  of  d- 
1-arabinose  and  recovered  4.5  grams  of  d-,  and  only  1.04  grams  of  1-arabinose  in 
the  urine.  On  the  other  hand  1-arabinose  becomes  converted  in  part  into  the 
dextro-form,  since  both  forms  appear  in  the  urine  when  only  one  is  given.  Xylose 
has  been  found  to  behave  in  general  like  arabinose.-' 

Since  all  writers  agree  that  10  to  50  per  cent,  of  administered  pentoses  are 
excreted  in  the  urine  even  when  given  per  os  in  very  small  quantities,  and  since 
pentoses  occur  in  many  foods  (plums,  cherries,  apples,  etc.)  or  result  from  the 

'*  Rhamnose  is  a  methyl  pentose,  representing  a  class  of  substances  closely 
related  to  the  jjentoscs. 

'»  Virchow's  Archiv.,  1892  (129),  401. 

i""  Festschr.  f.  E.  v.  Leyden,  1902  (2),  401. 

2'  Dcut.  Arch.  f.  klin.  Med.,  1897  (58),  523. 

"  Zeit.  f.  physiol.  Chem.,  1902  (35),  41. 

"  For  literature,  see  Neuberg,  "  Der  llaru  sowie  die  iibrigen  Aussclieiduiigen, 
etc."      (Springer,  Berlin,  1911),  1,  p.  370. 


PENTOSES  AM)  II  EX  OSES  657 

bacterial  ilccoiiiiKJsitiou  of  other  carhohyiliatcs,  it  is  inevitable  that  alimentary 
pentoaurias  should  oecasionaily  occur  in  nearly  every  normal  individual,  and,  a.s  a 
matter  of  fact,  most  normal  urines  give  reactions  which  indicate  the  ])resence  of 
some  pentose  (Cremer,  Funaro,  Cominotti).  Vice  versa,  one  may  ccMiclude  that 
very  little  jjcntose  normally  occurs  in  the  blood,  since  otherwise  more  of  it  would 
appear  in  the  normal  urine  than  does;  and  finally,  that  pentoses  must  play  but  a 
minor  role  in  the  general  metabolism  of  the  carbohydrates.  Therefore  it  is  highly 
imi)robable  that  during  the  breakdown  or  synthesis  of  glucose  in  the  body  the  hex- 
oses  split  to  any  extent  into  a  pentose  and  fornuUdehyde.  The  .same  holds  good 
for  the  behavior  of  hexoses  in  the  presence  of  alkali.  They  split  almost  exclu- 
sively into  chains  of  2,  3  and  4  carbon  atoms  (Xefj. 

Chronic  Pentosuria-* 
The  literature  contains  reports  of  some  30  cases  in  which  consid- 
erable quantities  of  pentose  have  been  excreted  steadily  in  the  urine 
regardless  of  the  character  or  quantity  of  the  food.  Even  during  a 
fast  the  quantity  excreted  has  remained  virtually  constant  in  some 
cases.  Outputs  as  high  as  36  grams  per  day  have  been  recorded. 
Such  quantities  of  pentose  could  not  be  introduced  into  the  body  from 
without  by  any  known  means  without  causing  pentosuria  of  marked 
degree.  Accordingly,  in  some  cases  there  is  cither  an  overproduction 
of  endogenous  pentose,  or  an  abnormal  entry  into  the  blood  stream 
of  pentose  which  is  normally  bound  in  the  tissues.  The  process  would 
then  be  analogous  to  that  in  which  lactose  from  the  mammary  glands 
occasionally  gains  access  to  the  general  circulation  and  appears  in  the 
urine.  This  conclusion  is  confirmed  by  the  work  of  Bial,  Blumenthal 
and  Tintemann,  who  found  that  certain  pentosurics  displayed  no  les- 
sened tolerance  for  administered  pentose.  The  origin  of  the  pentose 
is  unknown.  Nucleo-protein  of  cell  nuclei,  and  galactose  have  been 
suggested  as  possible  sources.  The  disease  has  been  found  in  differ- 
ent members  of  the  same  family  and  appears  to  be  a  harmless  anomaly. 
The  pentose  found  in  the  urine  in  cases  of  all  types  has  sometimes 
been  reported  as  optically  inactive,  sometimes  as  dextro-  or  levo- 
rotatory;  1-arabinose  (dextro-rotatory),  d-xjdose  and  d-xyloketose 
appear  to  have  been  identified.-^ 

HEXOSES 
Chemical  Introduction. — Structural  theory  demands  the  existence  of  32 
isomeric  hexo&e  sugars  of  the  formula  CeHijOg.  The  behavior  of  the  hexoses 
when  dissolved  in  very  dilute  alkali  makes  it  convenient  to  consider  them  in  four 
natural  series  of  eight  members  each.  Thus  one  series  comprises  the  8  hexoses 
whose  structural  formulae  appear  below.     This  may  be  called  the  d-glucose  series. 

(1)  (2)  (3)     ■  (4)  (5)  (6)  (7)  (8) 

CHO  CHO  CHoOH      CHO  CHO  CH5OH       CH-OH  CH-OH 

H-(4-0H  HO-i-H  60  H-i-OH  HO-t-H  60  H-COH      HO-C-H 

HO-(i-H  H0-(i-H       HO-c'^-H  K-6-0K       H-6-OH    H-t-OH  CO  tO 

H-(i-OH      H-(i-OH      H-(!^OH  H-(LoH  H-C-OH   H-C-OH    H-(!:-OH       H-i-OH 

H-i-OH      H-i-OH      H-ioH  H-i-OH       H-C-OH   H-C-OH    H-C-OH       H-C-OH 

CH2OH         CH2OH         CH20H      CH2OH  CHiOH      CHiOH       CH3OH  CHjOH 

d-pseudo 
d-glucose     d-mannose     d-fructose      d-allose        d-latose        fructose     a-d-glutose  ^-d-glutose 

2<See  Garrod.   ''Inborn  Errors  of  Metabolism,"   Oxford  Med.   Publ.,    1909; 
Lancet,  July,  1909. 

25  For  literature  see  ffiUer,  Jour.  Biol.  Chem.,  1917  (30).  129. 

42 


658  DIABETES 

There  is  also  an  1-glucose  series  in  which  the  members  are  the  mirror  images 
of  the  above.  There  is  a  third  series  comprising  d-galactose,  d-talose,  d-tagatose, 
1-sorbose,  1-idose,  1-gulose  and  alpha  and  beta  d-galtose;  and  a  fourth  series  whose 
relationship  to  the  d-galactose  series  is  the  same  as  that  of  the  1-glucose  to  the 
d-glucose  series.  Consideration  of  the  d-glucose  series  will  bring  out  the  prin- 
ciples common  to  all.  Examination  of  the  8  formulae  shows  that  numbers  1,  2, 
4  and  5  have  aldehyde  groups  (H — C  =0)  at  the  end  of  the  chain  and  are  hence 
aldohexoses.  Members  3  and  6  have  ketone  (C=0)  groups,  at  the  second  car- 
bon atom,  and  are  therefore  2-keto-hexoses,  while  numbers  7  and  8  having  ketone 
groups  at  the  third  carbon  atom  are  3-keto-hexoses.  Since  each  series  of  8  sugars 
has  a  like  number  of  the  different  types  there  are  in  all  16  aldohexoses,  eight  2- 
keto-  and  eight  3-keto-hexoses. 

It  had  long  been  known  that  if  a  solution  of  any  optically  active  sugar,  such 
as  d-glucose,  was  alkalinized  the  solution  gradually  lost  its  optical  activity.  It 
was  later  shown  by  Lobry  de  Bruyn  and  van  Ekenstein  that  a  solution  of  d- 
glucose  in  very  dilute  alkali  comes  to  contain  a  group  of  4  hexoses  in  dynamic 
chemical  equilibrium. ^'^  Nef^'  held  that  in  such  solutions  there  is  an  equili- 
brium of  at  least  eight  hexoses  as  above  depicted.  Any  one  of  these  sugars  when 
placed  in  alkali  reproduces  the  other  seven,  since  the  members  of  the  series  are 
reciprocally  convertible  one  into  another.  The  same  holds  good  for  the  members 
of  the  d-galactose  series  and  for  the  1-galactose  and  1-glucose  series.  But  the 
reciprocal  transformation  of  the  members  of  one  series,  such  as  d-glucose,  into  a 
hexose  of  another  series,  such  as  d-galactose,  occurs,  if  at  all,  only  to  a  minute 
degree,  because  such  transformation  involves  the  breaking  of  the  hexose  chain  into 
2,  3  and  4  carbon  fragments  with  subsequent  recombinations,  and  when  this  occurs 
irreversible  reactions  are  prone  to  intervene.  The  formation  of  lactic  acid  and 
the  saccharines  are  representative  of  these  irreversible  reactions.  (In  the  body, 
however,  glucose  may  be  converted  into  lactic  acid  in  the  muscles  and  elsewhere, 
whereas  in  diabetes  lactic  acid  can  be  converted  easily  back  into  glucose;  in  diabetes 
galatose  is  convertible  into  glucose,  etc.,  so  that  in  the  body  the  transformation 
of  hexoses  of  different  groups  one  into  another  offers  no  difficulty.) 

In  order  to  explain  the  effects  of  dilute  alkali  on  hexoses  just  described,  some 
conception  of  labile  intermediate  products  is  a  logical  necessity,  for  when  levulose 
changes  into  glucose  there  is  necessarily  some  intermediate  phase.  The  nature  of 
these  phases  has  been  the  subject  of  study  by  many  chemists,  and  this  study 
involves  always  the  question  of  sugar  dissociation. 

Sugars  are  weak  acids.  They  form  salts  with  metals,  and  Cohen-^  and  later 
Michaelis  and  Rona^"  have  determined  by  physico-chemical  methods  the  ioniza- 
tion constants  for  glucose  and  other  sugars.  Sugars  are  also  polyatomic  alcohols, 
and  either  aldehydes  or  ketones.  Sugar  chemistry  reverts  to  the  chemistry  of 
these  three  classes  of  compounds. 

A.  P.  Mathews'"  and  Michaelis  have  suggested  that  the  effect  of  alkali  on  a 
sugar  such  as  glucose  is  to  increase  enormously  the  concentration  of  the  glucose 
anion,  i.  e.,  KOH  leads  to  the  formation  of  K-glucosate  (see  Fig.  3,  p.  21),  which, 
being  the  combination  of  a  powerful  base  with  a  very  weak  acid,  has  a  high  elec- 
trolytic dissociation  constant.  These  anions  according  to  this  view  are  sub- 
ject to  cleavages  and  intramolecular  rearrangements.  Nef  also  hokls  that  the 
first  effect  of  the  alkali  {e.  g.,  KOH)  is  to  form  a  salt,  but  his  far  more  detailed 
conception  of  the  subsequent  changes  which  lead  to  reciprocal  transformations 
of  hexose  sugars  one  into  another,  involves  other  i)rinci]>les  which  represent  the 
outgrowth  of  his  earlier  work  on  the  properties  of  simpler  aldehydes  and  ketones. 

These  reciprocal  transformations  are  dependent,  according  to  Nef,  upon  the 
aldehydic  or  ketonic  character  of  the  sugar,  whereas  the  oxidative  phenomcnaand 
the  saccharinic  acid  formation  to  be  described  presently,  tlepend  upon  the  alcohol 
groups.  The  principles  involved  can  best  be  understood  if  we  first  consider  the 
behavior  of  a  simi)le  aldehyde  (acetaldehj^de)  and  a  simple  ketone  (acetone). 

26Rec.  trav.  chim.  de  Pays  Bas  (14),  158  and  203;  (15),  92;  (16),  257;  (19), 
1  and  10. 

"Liebig's  Annalen,  1907  (357),  294;  1910  (370),  1. 

28  Zcit.  f.  i)hysikal.  Chem.,  1901  (36),  09. 

29  Biochem.  Zeit.,  1912  (47),  447. 
3"  Jour.  Biol.  Chem.,  1909  (6),  1. 


CHEMISTRY  OF  THE  IIEXOSES  659 

Acetaldchyde  in  the  presence  of  water  forms  a  liydrato  (coiiiparal)lc  to  cliloral 
hydrate). 

H  H 

I 
CH3-C=0  +  HOHfiCH,— C—OH 

OH 

This  hydrate  possesses  ionizable  hydro(?en  in  its  OH  groups,  and  in  the  presence 
of  a  metallic  hydroxide,   MOH,  can  accordingly  form  a  salt  (comi)arahle  to  an 
H 

alcoholate) ;  thus :  CH3  -  C  -  OH, 

OM 

and  this  salt  being  highly  dissociable  falls  apart  into  MOH  and  a  "methylene 
enol "  (in  this  case  hydroxyethylidene) : 

H 

I  I 

CHs-C-OH^CHa-C-OH  +  MOH 

OM 

(Herein  lies  the  point  of  departure  of  Nef's  view  from  the  foregoing.)  This 
methylene  enol  then  rearranges  to  form  the  "olefine  dienol"  CH2  =  CHOH  (in  this 
case  vinyl  alcohol).  Ketones,  on  the  other  hand,  form  the  olefine  dienol  directly 
without  forming  the  methylene  enol.  In  the  case  of  KOH  and  acetone  (dimethyl 
ketone)  there  is  the  same  formation  of  the  hydrate  followed  by  salt  formation  and 
the  loss  of  KOH,  but  the  latter  does  not  all  come  from  one  C  atom  as  it  does 
when  split  out  of  aldehydes,  thus : 

OH  OK 

I  I 

CHs-C-CHa  +  KOH^CHj-C-CH,,  +  HjO^  CH3-C-CH2 
I 
OH  OH  OH 

(hydrate  of  acetone) 

In  a  manner  entirely  analogous  to  what  occurs  in  the  simple  aldehydes  and 
ketones,  the  two  aldohexoses,  glucose  and  mannose,  and  the  ketohexose  levulose, 
can  form  one  and  the  same  enol  molecule.  And  vice  versa  this  enol  molecule  may 
open  its  double  bond  in  two  ways  as  shown  below  (  (a),  (b)  and  (c)  )  and  the 
dissociated  molecules. 


(a) 
OH 

(c) 
OH 

(b) 
OH 

(d) 

OH 

1 

(e) 
H 

0- 

I 
-C— 

1 

H- 

-C 

II 

C— OH 

H- 

-C- 

H— C 

II 
C- 

H- 

-C— OH 

HO- 

-c— 

2 

-C— OH 

-OH 

C— OH 

HO- 

-C— H 

3 

HO- 

-C— H 

HO- 

-C— H 

1 

H— C- 

1 

-OH 

H- 

-C— OH 

1 

H- 

-C— 0H4 

H- 

-C— OH 

1 

H- 

-C— OH 

1 
H— C- 

1 

-OH 

H- 

-C— OH 

H- 

-C— OH  5 

H- 

-C— OH 

H- 

-C— OH 

H— C- 

-OH 

H- 

-C— OH 

H- 

-C— OH 
H 

6 

H- 

-C— OH 
H 

H- 

-C— OH 

1 
H 

H— C- 

1 

H 

-OH 

H- 

-C— OH 
H 

Enol  molecule 
(a,  1,  2  d-glucose  olefine  dienol) 

(a)  and  (b)  will  be  in  dynamic  equilibrium  with  the  enol  (c).     Now  if  H  and 
OH  are  again  taken  on  by  (a)  and  (b)  this  assumption  of  the  elements  of  water 


660 


DIABETES 


can  take  place  in  three  different  ways,  to  regenerate  d-levulose,  d-mannose  and 
d-glucose.     Thus  if  in  the  case  of  (a),  OH  is  added  to  carbon  atom  number  one 
OH 

this  will  form  the  group  H — C — OH  which  represents  a  hydrated  aldehyde  group 

and  will  lose  water  to  become  CHO.  Then  H  going  to  the  second  C  atom  com- 
pletes the  formula  of  d-mannose.  In  a  similar  way  (b)  can  form  d-glucose. 
But  if  the  OH  went  to  the  second  carbon  atom  this  group  would  thereby  become 

a  hydrated  ketone  similar  to  the  hydrate  of  acetone  and  lose  water  to  form  C  =  O, 

while  H  going  to  the  end  carbon  atom  would  complete  the  formula  of  d-levulose. 
In  a  manner  entirely  analogous  there  is  an  enol  molecule  which  is  common  to  d- 
allose,  d-lactose  and  d-pseudo  fructose  (see  (d)  above). 

It  will  be  noticed  that  each  of  these  enols  is  in  equilibrium  with  a  2-keto- 
hexose  and  two  aldo-hexoses.  Now  these  2-keto-hexoses,  d-fructose  and  d-pseudo 
fructose,  in  accordance  with  general  ketone  behavior,  are  capable  of  yielding  .an- 
other common  enol,  i.  e.,  a  2-3  enol  (with  the  double  bond  between  the  second  and 
third  carbon  atoms)  as  represented  at  (e)  and  this  2-3  enol  (by  a  process  like 
that  just  detailed  for  the  1-2  enols)  can  account  for  the  formation  of  the  two 
3-keto-hexoses  whose  formulae  are  given  above.  The  same  general  principles  hold 
for  each  of  the  series  of  hexoses  (For  further  elaboration  of  the  theory  see  Xef's 
original  papers.)  A  similar  use  of  enol  molecules  in  this  connection  is  made  by 
Neuberg. 

It  remains  now  to  point  out  that  if  to  a  simple  aqueous  solution  of  sugar, 
oxygen  be  supplied  in  the  form  of  air  or  H2O2,  no  oxidation  occurs.  But  if  the 
solution  be  alkalinized  then  the  sugar  is  readily  burned.  In  the  absence  of  oxygen 
and  the  presence  of  alkali  somewhat  stronger  than  that  found  most  favorable 
for  the  reciprocal  transformation  above  detailed,  there  occur  certain  irreversible 
reactions  such  as  the  formation  of  lactic  acid  and  the  so-called  saccharines.  When 
an  alkaline  sugar  solution  is  treated  with  oxygen  it  yields  CO2,  HoO,  and  formic, 
glycoUic,  glyceric  and  certain  trihydroxy-butyric  and  hexonic  acids,  depending 
on  the  sugar  used.  Without  oxygen,  or  with  too  little  oxygen,  lactic  acid  and  the 
saccharinic  acids  make  their  appearance  (cf.  the  formation  of  lactic  acid).  The 
explanation  of  these  phenomena  rests  in  the  conception  thai  alkali  increases  the 
dissociation  of  sugar,  and  that  the  dissociated  fragments  burn  or  rearrange  depend- 
ing upon  the  conditions  of  the  experiment. 

i-  In  this  connection,  according  to  Nef,  we  are  dealing  with  the  alcohol  groups 
of  the  sugars  and  may  advantageously  turn  for  a  moment  to  the  properties  of 
methyl  alcohol.     This  substance  consists  under  ordinary  circumstances  of  a  great 


( 

31ucose 

Glucose  ion 

(1) 

0 

II 

C— H 

1 

(2) 
(— ) 
0 

C— H 

H- 

-C— OH 

H- 

-C— 0—  - 

OH- 

-C— H 

OH- 

-C— H 

H- 

-C— OH 

H- 

-C— OH 

H- 

-C— OH 

H- 

-C— OH 

H- 

-C— OH 

1 

H- 

-C— OH 

1 
H 

H 

-H 


K-glucosate 

(3) 

Methylene 
particle 

(4) 

0 

0 

C— H 

H— C— OK 

- 

-c— 

OH— C— H 

HO- 

-C— H+KOH 

H— C— OH 

H- 

-C— OH 

1 

H— C— OH 

1 

H- 

-C— OH 

H— C— OH 

H- 

-C— OH 

H 


H 


preponderance  of  undissociated  molecules  in  d3'namic  equilibrium,  with  a  very 
minute  quantity  of  dissociated  methylene  CHsOH?:^  CH2  -|-  H2O.     The  primary 


GALACTOSE  661 

effect  of  iilkali  (KOII)  is  to  form  a  salt,  C^IIj — OK  (or  l\-incthylatc),  wliicli  heing 
highly  dissociable  breaks  down  to  give  CHj  and   i\()H.     The  proportion  of  free 

II 
methylene  is  therel)y  enormously  increased.  What  then  befalls  the  methylene 
will  depend  on  the  amount  of  oxygen  i)resent  and  on  the  various  other  factors 
which  enter  into  the  conditions  of  the  experiment.  These  general  principles  arc 
applicable  directly  to  the  polyatomic  alcohols— the  hexo.ses  and  other  sugars — as 
shown  on  prcccdinp;  page. 

In  the  presence  of  sufHcient  oxygen  the  methylene  particle  takes  on  oxygen 
to  form  first  an  osone.  In  the  alisence  of  oxygen  it  undergoes  intramolecular 
rearrangements,  the  details  of  which  need  not  here  l)e  entered  into.  It  is  these 
which  gives  rise  to  the  (j-carbon  acids  known  as  the  saccharines  or  saccliarinic 
acids. 

GALACTOSE 

A  normal  individual  weighing  75  kilos  may  eat  about  50  grams  of 
galactose  and  show  but  a  trace  of  melituria.  More  than  this  is  likely 
to  cause  the  presence  of  measurable  amounts  of  galactose  in  the 
urine,  the  alimentary  tolerance  lintit  for  this  sugar  being  therefore 
about  0.6  to  0.8  grams  per  kilogram  of  body  weight.  We  have  no 
direct  data  concerning  the  time  within  which  50  grams  of  galactose 
are  absorbed  by  a  man  of  average  weight.  When  given  intravenously 
at  uniform  rates,  unchanged  galactose  appears  in  the  urine  of  dogs 
receiving  slightly  more  than  0.1  per  kilo  per  hour.  The  tolerance 
for  galactose  appears  to  be  lessened  in  phosphorus  poisoning  and  in 
many  other  conditions  which  cause  apparent  parenchj^matous  changes 
in  the  liver,  so  that  after  administration  of  50  grams  of  gakictose  by 
mouth,  as  much  as  10  to  12  grams  may  be  excreted  in  the  urine 
(Bauer).  On  the  other  hand,  ligation  of  the  common  duct  does  not 
lessen  the  tolerance  for  galactose  in  rabbits  (Reiss  and  Jehn,  Hierose) 
so  that  the  lowered  tolerance  following  phosphorus  administration  ap- 
pears to  be  independent  of  the  disturbed  biliary  function.  Infants- 
suffering  from  gastro-enteritis  may  show  alimentary  lactosuria,  and 
along  with  the  lactose  some  of  its  constituent  galactose  may  appear  in 
the  urine.  The  question  thus  naturally  arises  as  to  whether  the  low- 
ered tolerance  for  galactose  in  phosphorus  poisoning  and  other  liver  dis- 
eases may  not  be  due  to  an  increased  permeabilit}-  of  the  intestinal  wall,, 
or  to  changes  elsewhere  in  the  body  besides  the  liver,  ^^'orner  found 
that  galactose  injected  directly  into  the  portal  vein  was  handled  by 
healthy  and  phosphorized  rabbits  in  the  same  relative  jiroportions  as 
when  given  to  these  animals  by  mouth,  thus  apparently  exchuhng  the 
bowel  as  a  contributer  to  the  decreased  tolerance.  It  is  unlikf^Iy  also- 
that  the  kidneys  in  phosphorized  animals  were  rendered  abnormally- 
permeable  for  galactose,  since  when  the  kidneys  alone  are  phosphorized 
without  affecting  the  liver,  the  excretion  of  galactose  after  adminis- 
tration by  mouth  or  into  a  vein  has  been  retarded  rather  than  hastened. 
These  principles  have  become  incorporated  in  a  clinical  test  for  dis- 
ease of  the  hepatic  parenchyma.  When  galactose  is  administered  to  a 
fully  diabetic  animal  it  is  capable  of  being  converted  quantitatively 
into  glucose.     Existing  data  indicate  that  galactose,  like  diose  and 


662  DIABETES 

glyceric  aldehyde,  is  chiefly  converted  into  glucose  before  further  util- 
ization, and  that  this  process  is  carried  out  mainly  in  the  liver  and 
bowel  wall.     The  literature  of  the  subject  is  given  below. ^^ 

LEVULOSE  (FRUCTOSE) 

The  group  of  eight  sugars  formed  by  levulose  in  the  presence  of 
alkali  includes  glucose,  and  any  member  of  this  group  will  produce 
all  the  others.  Then,  in  cases  of  glycosuria  with  alkaline  urine 
(whether  physiological  or  due  to  medication  or  to  bacterial  decompo- 
sition), levulose  might  be  expected  to  occur  along  with  glucose.  ^lay 
and  Koenigsfeld  have  reported  instances  of  this  "urinogenous  levu- 
losuria."     Magnus-Levy  doubts  the  correctness  of  these  observations. 

Alimentary  Levulosuria. — The  tolerance  of  a  normal  body  for  levu- 
lose given  per  os  is  variable.  Doses  of  50  to  70  gm.  in  man  cause  as  a 
rule  no  levulosuria,  but  more  is  likely  to  do  so.  Animals  in  which 
the  liver  parenchyma  has  been  damaged  by  phosphorus  are  said  to 
have  a  lower  tolerance.  In  many  other  diseases  of  the  liver  the  same 
holds  true,  and  H.  Strauss  believed  this  fact  could  be  made  the  basis 
of  a  clinical  test  for  liver  function.  Naunyn,  however,  emphasizes 
the  fact  that  in  certain  cases  of  cirrhosis  of  the  liver  with  collateral 
anastomoses  between  the  portal  vein  and  vena  cava,  there  is  also  a 
lessened  tolerance  for  levulose  given  by  mouth,  owing  to  the  fact  that 
levulose  then  enters  the  general  circulation  without  having  entered 
the  liver.  As  far  back  as  1871  Eichhorst  showed  that  levulose  intro- 
duced per  rectum,  i.  e.,  where  it  will  presumably  enter  an  hemor- 
rhoidal vein  after  resorption,  is  more  likely  to  cause  alimentary 
levulosuria  than  when  swallowed,  because  of  the  extra-hepatic  an- 
astomoses between  the  hemorrhoidal  veins  and  the  vena  cava.  As  in 
the  case  of  diose,  glyceric  aldehyde  and  galactose,  intravenous  injection 
of  levulose  produces  levulosuria  in  dogs  when  the  rate  of  injection  is 
between  0.1  and  0.2  gram  per  kilo  per  hour.  These  facts  support  the 
belief  that  the  liver  plays  the  same  important  part  in  "assimilating" 
levulose  as  with  other  sugars. 

Spontaneous  Alwientary  Levulosuria,  i.  e.,  the  appearance  of  lev- 
ulose in  the  urine  from  such  small  quantities  of  levulose  as  occur 
naturally  in  the  food,  has  been  demonstrated  in  eight  cases.  In  five 
of  these  levulose  appears  to  have  been  the  only  sugar  present.  These 
persons  showed  a  decreased  tolerance  for  ingested  levulose  and  ceased 
passing  the  sugar  when  the  diet  was  carbohydrate-free.  The  tend- 
ency of  thought  would  be  to  look  for  the  cause  of  such  phenomena 
in  a  disturbed  hepatic  function. 

"  Bauer,  Deut.  med.  Woch.,  1908  (35),  1505;  Reiss  u.  Jehn.  Deut.  Arch.  f. 
Min.  Med.,  1912  (108),  187;  Roubitscheck,  Deut.  Arch.  f.  klin.  Med.,  1912  (108), 
225;  Naunyn,  "Beitrage  zur  Lehre  von  Ikterus,  etc.,"  Reichcrt-Dubrissches 
Archiv.  ftir  Anatomic,  1869,  p.  579;  Schopffer,  Arch.  f.  exp.  Path.  u.  Phamo.,  1873 
(1),  73. 


LEVULOSE  AS  I)  LACTOSE  m'i 

It  is  interesting  to  note  that  of  the  above-mentioned  five  cases  of 
pure  levulosuria,  two  showed  a  lessened  tolerance  for  glucose,  and  one 
symptoms  of  dispituitarism,  one  developed  during  the  puerfjcrium, 
and  one  had  an  endocarditis;  i.  e.,  four  out  of  live  had  evidence  of 
derangements  of  the  endocrinous  glands.  The  literature  has  been 
reviewed  and  a  case  reported  by  Strouse  and  Friedman. ^'■^ 

Mixed  Levulosuria,  or  the  occurrence  of  levulose  along  with  glucose 
in  severe  cases  of  diabetes,  is  said  by  some  to  be  a  common  event.  In 
view  of  the  great  frequency  of  combined  liver  and  pancreatic  changes 
found  at  autopsy  in  diabetic  cases,  and  in  view  also  of  the  frequent 
occurrence  in  diabetes  of  signs  which  point  to  disturbances  of  other 
glands  with  internal  secretion  besides  the  pancreas,  this  would  har- 
monize well  with  the  view  just  given. 

Spontaneous  or  Idiopathic  Levulosuria,  having  a  character  similar 
to  chronic  pentosuria,  and  running  a  steady  course  uninfluenced  by 
diet,  has  been  reported  in  one  case  by  Rosin.  In  this  instance  the 
tolerance  for  glucose  was  also  diminished. 

POLYSACCHARIDES 

Closely  related  to  these  meliturias  are  the  forms  in  which  the  poly- 
saccharides,— lactose,  maltose  and  saccharose, — are  the  sugars  con- 
cerned. 

Lactosuria: — -When  2  to  3  grams  of  lactose  per  kilogram  of  body 
weight  are  given  in  pure  form  by  mouth  to  a  healthy  adult  dog  or 
man — alirnentary  lactosuria  generally  occurs.  Another  form  of  lacto- 
suria is  that  seen  in  lactating  women.  In  these  cases  the  lactose  gains 
access  to  the  general  circulation  from  milk  stasis  in  the  breast.  Yet 
another  form,  the  lactosuria  in  children,  having  gastro-intestinal  dis- 
eases, has  its  origin  in  the  lactose  of  the  milk  or  artificial  food.  In 
these  cases  lactosuria  may  develop  after  the  ingestion  of  lactose,  in 
quantity  and  form^^  incapable  of  causing  it  in  a  healthy  child.  The 
tolerance  for  lactose  is  most  strikingly  decreased  in  so-called  "intoxi- 
cation" (Finkelstein)  in  which  lactosuria  may  follow  ingestion  of 
0.4-0.5  g.  per  kilo  of  body  weight.  (Grosz  places  the  assimilation  limit 
for  healthy  sucklings  at  8.6  g.  per  kilo.)  This  might  be  explained  in 
two  or  more  ways.  The  lactase  in  the  bowel  might  be  deficient  and 
permit  unhydrolyzed  sugar  of  milk  to  accumulate  in  abnormal  con- 
centration in  the  lower  bowel,  and  then  be  absorbed  unsplit ;  or,  as 
seems  more  probable,  the  bowel  wall — because  of  ulcers  or  simple  in- 
flammatory changes — ^  might  become  abnormally  permeable.  The 
intravenous  tolerance  limit  for  lactose  approaches  zero.  During  pro- 
longed intravenous  injections  of  lactose  into  dogs  at  the  rate  of  2  gm. 

32  Arch.  Int.  Med.,  1912  (9),  99. 

'3  Pure  aqueous  solutions  of  sugar  differ  in  the  rate  of  absorption  from  those 
"n  which  the  sugar  is  incorporated  in  heterogeneous  mixtures. 


664  DIABETES 

per  kilo  per  hour,  lactose  was  excreted  at  the  rate  of  injection  during 
the  fourth  hour  and  the  following  four  hours. ^^ 

Leopold  and  Reuss  reported  that  when  1  gram  of  lactose  was 
injected  subcutaneously  into  a  dog  or  infant,  exactly  1  gram  reap- 
peared in  the  urine;  but  that  if  the  injections  were  made  daily,  the 
quantity  excreted  fell  little  by  little  and  finally  became  zero.  Helm- 
holz  and  Woodyatt  have  repeated  this  experiment  in  dogs,  and  found 
that  at  first  the  gram  injected  might  reappear  in  the  urine  as  stated. 
Sometimes,  however,  the  occurrence  of  an  increase  in  the  reducing 
power  of  the  urine  above  the  figure  representing  1  gram  of  lactose 
was  noted.  This  suggested  a  splitting  of  the  lactose  into  glucose 
and  galactose.  Nor  could  they  obtain  more  than  a  temporary  disap- 
pearance of  the  sugar  following  subsequent  injections,  even  when 
carried  on  for  weeks — such  as  Leopold  and  Reusse  reported.  The 
point  of  chief  interest  in  these  experiments  is  that  the  apparently  in- 
creased hydrolysis  of  lactose  developing  with  successive  doses  resem- 
bles a  reaction  of  immunity,  with  a  substance  of  known  chemical 
composition  as  the  antigen.  But  it  is  possible  that  the  successive  in- 
jections simply  result  in  a  lessened  excretion  of  the  lactose  by  the  kid- 
neys. Abderhalden  and  his  co-workers  reported  that  the  serum  of 
animals  similarly  treated  develops  an  increased  power  to  split  the  di- 
saccharide  employed,  as  determined  by  means  of  the  polariscope. 
These  experiments  were  paralleled  with  cane  sugar  (saccharose)  and 
with  di-,  tri-,  and  higher  peptids.  Other  observers  have  failed  to  cor- 
roborate these  findings. 

Saccharosuria  (cane  sugar  in  the  urine)  occurs  under  conditions 
quite  similar  to  those  mentioned  for  lactose,  except  that  there  is  no 
saccharosuria  corresponding  to  the  lactosuria  of  women. 

Maltosuria  has  often  been  reported,  but  the  chemical  detection  of 
this  sugar  is  uncertain. 

Other  polysaccharoses,  such  as  isomaltose,  glycogen,  etc.,  have  been 
thought  by  some  writers,  to  occur  in  the  urine. 

GLYCOSURIAS 

Glucose  is  the  sugar  which  enters  into  the  normal  glycogen  and 
forms  the  bulk  of  the  body  sugar.  Glycosurias  are  naturally  the 
most  important  of  the  meliturias. 

(1)  Alimentary  glycosuria,  e  saccharo.  Not  infrequently  it  is  im- 
possible to  make  a  healthy  man  eat  and  retain  sufficient  glucose  to 
cause  glycosuria,  and  it  would  be  hard  to  define  an  increased  glucose 
tolerance.  This  statement  is  corroborated  by  the  studies  of  Taylor 
and  Hulton^''  on  man.  As  indicated  before,  an  increased  supplj^  of 
glucose  to  the  body  may  increase  the  urinary  sugar  and  as  Benedict 
et  al.  have  stated,  there  may  be  no  sharp  line  of  division  between  those 

'■•  Unpublished  experiments  by  W.  D.  Sansum. 
»  Jour.  Biol.  Chem.,  1916  (25),  173. 


chcaiigcs  and  a  {j;n)ss  {rlycosuria.  In  do^s  wciKliin^  10  kilos  llu-  niaxi- 
niuni  rate  of  glucose  absorption  is  apparently  reached  with  doses  of 
50  grams  and  perhaps  less.  Larger  doses  do  not  further  increase  the 
rate  of  aljsorption.  This  rate  may  be  l.S  gram  per  kilo  jjcr  hour.  If 
with  this  rate  of  absorption  the  rate  of  utilization  in  the  bow(;l  wall 
and  liver  is  0.9  gram  per  kilo  per  hour,  or  less,  glucose  will  enter  the 
systemic  blood  at  the  rate  of  0.9  gm.  per  kilo  per  hour,  or  more,  and 
this  will  normally  cause  a  gross  glycosuria.  The  physiological  state  of 
the  liver  and  the  rate  of  sugar  absorption  are  factors  of  importance 
in  determining  alimentary  glycosuria.  CUycosuria  following  the  in- 
gestion of  starch  alone — alimentary  glycosuria  ex  amylo  was  said  not  to 
occur  in  healthy  individuals,  but  the  feeding  of  large  quantities  of 
starch  increases  the  urinarj-  sugar  and  if  the  tests  used  for  its  detection 
are  of  sufficient  delicacy  the  increase  is  measurable.  With  a  sufhci- 
ently  small  urinary  volume  ordinary  tests  may  detect  the  extra  sugar. 

(2)  Glycosurias  which  depend  upon  the  discharge  of  sugar  from 
stored  glycogen.  These  may  be  due  to  the  action  of  (a)  nerves,  (b) 
drugs,  (c)  the  so-called  internal  secretions. 

(a)  Claude  Bernard's  piqure,  or  puncture  of  the  floor  of  the 
fourth  ventricle  between  the  points  of  origin  of  the  eighth  and  tenth 
pairs  of  nerves,  causes  a  glycosuria  which  ceases  when  the  glycogen 
of  the  liver  is  reduced  to  a  low  percentage.  Following  this  operation 
the  blood  is  found  to  contain  an  excess  of  sugar  (hyperglycemia)  to 
which  the  glycosuria  is  immediately  due.  If  the  vagus  nerve  is  cut 
stimulation  of  the  central  end  has  a  similar  effect,  so  that  the  vagus 
is  said  to  carry  the  afferent  impulse  to  the  center  in  the  calamus 
scriptorius.  By  severing  different  portions  of  the  nervous  system 
and  stimulating  the  cut  surfaces,  the  path  of  the  efferent  impulse  has 
been  traced  from  the  glycogenic  center  through  the  cord  to  the  upper 
thoracic  spinal  roots,  by  the  rami  communicantes  to  the  inferior  cer- 
vical and  superior  thoracic  ganglion,  thence  via  the  splanchnic  nerves 
to  the  liver.  This  center  and  nervous  arc  form  probably  an  im- 
portant link  in  the  mechanism  for  regulating  the  quantity  of  sugar 
in  the  blood.  Nervous  glycosurias  having  the  same  mechanism  as 
"la  piqtire"  occur  in  a  great  variety  of  conditions  associated  with 
insult  to  the  nervous  system,  e.  g.,  commotio  cerebri,  brain  tumor, 
tabes,  meningitis,  severe  mental  shock,  etc.  How  a  splanchnic  impulse 
operates  to  cause  increased  hydrolysis  of  glycogen  is  unsettled.  Gly- 
cogen hydrolyzes  outside  the  body  under  the  influence  of  acids,  i.  e., 
of  H  ions  or  plus  charges  of  electricity,  and  a  nerve  impulse  might 
theoretically  operate  directly,  or,  as  McLeod  has  suggested,  through 
an  increase  of  glycogenase  in  the  liver;  or  as  held  by  the  von  Noorden 
school,  by  causing  an  increased  section  of  epinephrine — since  piqure 
glycosuria  is  said  not  to  occur  in  animals  deprived  of  the  adrenals 
(Mayer,  Kahn,  Nishi)  or  after  section  of  the  left  splanchnic  nerve, 
which  supplies  both  adrenals,     (b)  Similar  phenomena  occur  in  as- 


666  DIABETES 

phyxia  (carbonic  and  lactic  acid  accumulation),  and  when  acids  are 
directly  administered;  also  after  the  administration  of  certain  drugs 
whose  effects,  including  lactic  and  carbonic  acid  accumulation  in  the 
body  fluids,  closely  parallel  those  of  a  deficient  oxygen  supply  (phos- 
phorus, carbon  monoxide,  chloroform,  hydrazine,  arsenic,  etc.). 
Certain  other  drugs,  such  as  curare,  strychnia,  etc.,  may  interfere 
with  respiratory  movements  and  so  cause  glycosuria  by  secondary 
asphyxia;  although  other  drugs,  of  which  there  are  many,  maj''  op- 
erate to  cause  glycosuria  in  any  of  the  ways  by  which  glycosuria  can 
be  produced. ^^ 

(c)  The  ductless  gland  extracts  which  produce  glycosuria  include 
those  of  the  adrenal,  thyroid  and  hypophysis.  Epinephrine  has  been 
discussed  in  another  place,  and  the  reasons  are  there  developed  for  the 
belief  that  the  glycosuria  it  causes  is  due  to  a  mobilization  of  sugar 
from  glycogen,  which  leads  to  hyperglycemia.  Ringer^"  showed  that 
when  an  animal  is  fully  phlorhizinized  the  subcutaneous  injection  of 
epinephrine  causes  no  additional  output  of  sugar  nor  alteration  of  the 
G  :  N  ratio,  a  fact  which  has  been  confirmed  by  Sansum  and  Woody- 
att — thus  proving  that  epinephrine  has  no  power  to  intensify  a  diabetes 
which  is  already  at  the  point  which  is  called  complete.  Lusk^^  also 
showed  by  respiration  experiments  the  correctness  of  this  interpreta- 
tion. Eppinger,  Falta  and  Rudinger^^  stated  that  epinephrine  inten- 
sifies pancreas  diabetes,  and  used  this  observation  in  support  of  their 
idea  that  epinephrine,  like  thyroid  extract,  exerts  in  the  liver  a  sugar- 
mobilizing  and  sugar-building  effect,  antagonistic  to  the  action  of  the 
pancreas,  which,  according  to  the  doctrine  of  the  von  Noorden  school, 
checks  the  formation  of  sugar  from  glycogen  and  also  from  protein 
and  fat.  But  in  their  work  there  has  been  no  adequate  proof  that  be- 
fore giving  the  epinephrine  the  pancreas  diabetes  was  as  complete  as  a 
pancreas  diabetes  can  be,  or  that  the  increased  intensity  of  the  dia- 
betes was  any  greater  than  could  have  been  explained  bj'  a  discharge 
of  sugar  from  glycogen.  The  power  of  pituitary  extracts  to  produce 
glycosuria  is  likewise  ascribable  to  their  effects  on  glycogen. 

PHLORHIZIN  DIABETES" 

Phlorhizin  was  obtained  by  alcoholic  extraction  of  the  bark  and 
roots  of  apple,  pear,  plum  and  cherry  trees  by  L.  de  Koninck  in  1835. 
Its  glucosidic  character  was  established  by  Stas,  who  found  that  it 

'^  The  production  of  glycosuria  by  a  given  drug  should  not  be  confused  with 
excretion  of  paired  glycuronic  acid  compounds,  such  as  occurs  after  the  ad- 
ministration of  many  aldehydes,  ketones,  alcohols  and  phenols.  The  reducing 
power  in  these  cases  is  not  due  to  glucose  but  to  its  oxidation  product, 
COOH— (CH0H)4— COH. 

"  Jour.  Exper.  Med.,  1910  (12),  105. 

38  Arch.  Int.  Med.,  1914  (13),  673. 

SB  Zeit.  f.  klin.  Med.,  1908  (66),  1;  1909  (67),  380. 

*"  For  a  treatise  of  the  whole  subject  of  ])hlc)rhizin  glycosuria,  with  bibliog- 
raphy, see  the  monograph  by  Lusk  (Phlorhizin  (Uykosurie,  Ergcb.  der  Physiol., 
1912  (13),  315),  free  use  of  which  has  been  made  in  the  following. 


PHLORHIZIN  DIABETES  (107 

could  be  split  into  glucose  ('' phlorose")  and  u  subytanee  (phlorctin) 
which  by  acid  hydrolysis  yielded  phloroglucin  and  an  acid  (phlorctinic 
acid).  It  was  not  until  18SC  that  von  Mcrin^^  puhjislied  his  first 
experiments  upon  its  physiolofj;ic  action. 

While  phlorhizin  causes  glycosuria  wiicn  taken  by  mouth,  its  great- 
est effect  is  obtained  by  subcutaneous  injection.  One  gram  of 
phlorhizin  triturated  in  5  to  15  c.c.  of  olive  oil,  or  in  20  per 
cent,  alcohol,  and  injected  subcutaneously  once  every  24  hours, 
will  maintain  the  maximum  glycosuria  which  can  be  produced 
in  a  dog  of  10  kilogrammes.  Phlorhizin  is  mostly  (80-90  per  cent.) 
excreted  in  the  urine.  It  is  soluble  in  ether,  optically  active,  and  gives 
a  garnet  coloration  with  ferric  chloride,  so  that  it  interferes  with  the 
polariscopic  tests  for  )3-hydroxybutyric  acid  in  the  urine,  and  masks 
the  Gerhardt  reaction  for  aceto-acetic  acid. 

Phlorhizin  causes  glycosuria  in  frogs  and  other  cold-blooded  ani- 
mals, as  well  as  in  warm-blooded  forms  in  general,  including  birds. 
That  geese  show  glycosuria  with  phlorhizin  (von  Mering,  Thiel)  is 
important,  because  birds  do  not  pass  sugar  in  the  urine  when  op- 
erations are  performed  upon  them  which  do  cause  a  definite  excess 
of  blood  sugar  (pancreatectomy,  Minkowski).  Phlorhizin  causes 
glycosuria  in  birds — hyperglycemia  does  not.  Hence  phlorhizin  does 
not  cause  glycosuria  by  producing  hyperglycemia.  In  harmony  with 
this  syllogism  are  the  data  obtained  by  Minkowski,  Levene,  von 
Czylharz  and  Schlesinger,  Lewandowsky,  Lepine,  Porcher,  Junkers- 
dorf,  Erlandsen,  Frank  and  Isaac — all  of  whom  have  found  the  blood 
sugar  concentration  in  phlorhizinized  animals  low  (0.065  per  cent.; 
0.072  per  cent.;  0.012  per  cent.,  etc.).  Conflicting  results  have  also 
been  published,  but  the  methods  emploj'ed  in  these  ifistances  have  not 
usually  been  beyond  criticism  (Pavy,  Biedle  and  Kolisch).  Even 
after  ligation  of  the  renal  vessels  or  bilateral  nephrectomy,  no  hyper- 
glj^cemia  has  been  demonstrated  in  phlorizinized  animals,  whereas  if 
phlorhizin  acted  by  liberating  sugar  from  glycogen  reserves  in  the 
liver  and  elsewhere,  or  from  any  source  distant  from  the  kidneys, 
hyperglycemia  might  be  expected.  In  view  of  these  facts  von  Mering 
himself  interpreted  the  action  of  phlorhizin  as  a  kidney  diabetes. 

Zuntz  injected  phlorhizin  directly  into  one  renal  artery  and  col- 
lected the  urine  from  each  kidney  separately.  The  kidney  on  the 
injected  side  almost  at  once  secreted  saccharine  urine,  and  the  other 
kidney  secreted  sugar  only  after  the  lapse  of  minutes.  This  experi- 
ment has  been  successfully  repeated  by  others,  and  seems  to  prove 
that  phlorhizin  can  cause  glycosuria  by  acting  directly  on  the  kid- 
neys. The  many  experiments  which  have  been  made  to  determine 
the  relative  blood  sugar  content  of  the  renal  artery  and  vein  during 
phlorhizin  glycosuria,  add  little  to  tliis  subject. 

The  questions  arise:  Are  the  kidney  cells  the  only  structures  which 
are  directly  affected  by  phlorhizin?  and,  What  is  the  exact  nature  of  the 
phlorhizin  effect? 


668  DIABETES 

Levene  collected  the  bile  of  phlorhizinized  dogs  and  found  that  it 
exhibited  reducing  power  after  the  phlorhizin  injection,  but  not 
before.  Ray,  McDermott  and  Lusk  failed  to  find  similar  properties 
in  vomited  bile  from  phlorhizinized  dogs.  Brauer  repeated Levene's 
work — -using  a  different  method  in  that  he  cleared  the  bile  with  lead 
acetate  prior  to  making  the  sugar  tests,  and  then  found  no  reducing 
substance.  Woodyatt  obtained  results  like  Levene's  but  found  later 
no  reaction  for  sugar  when  the  bile  was  cleared  in  the  way  Brauer 
recommended.  Still,  in  the  native  state  it  yields  characteristic  crj^s- 
tals  of  an  osazon,  and  ferments  with  yeast  after,  but  not  before 
phlorhizinization.  Karl  Grube  perfused  tortoise  livers  with  salt 
solution  containing  phlorhizin  and  was  able  to  cause  more  rapid 
deglycogenation  than  when  the  same  salt  solution  minus  phlorhizin 
was  used  in  control  experiments.  Now  Ray,  McDermott  and  Lusk, 
in  working  with  bile  which  had  been  in  the  alimentary  tract,  used 
material  that  had  had  time  to  lose  its  sugar  by  resorption.  Brauer's 
clearing  method  may  take  out  a  trace  of  sugar  even  if  present  origi- 
nally, and  it  must  be  said  that  there  is  some  evidence  favoring  the 
idea  that  phlorhizin  acts  in  the  liver,  although  much  less  strongly 
than  in  the  kidneys.  Attempts  have  also  been  made  to  demonstrate 
a  direct  action  of  phlorhizin  on  the  mammary  (Cornevins)  and  sweat 
glands  (Delmare).  Cornevins'  positive  findings  were  not  confirmed 
by  Cremer  and  Porcher,  whereas  Delmare 's  work  has  not  been  re- 
peated. But  R.  Pearce,  working  with  a  blood-sugar  method,  found  an 
increase  of  sugar  in  the  pancreatic  juice,  and  Underbill  has  brought 
further  evidence  in  support  of  a  general  action.  M.  H.  Fischer  had 
some  nephrectomized  frogs,  which  are  able  to  live  indefinitely  in  water 
because  they  excrete  through  the  skin.  With  the  writer  some  of 
these  frogs  were  injected  with  phlorhizin  into  the  dorsal  lymph  sac, 
and  sugar  was  found  next  day  in  the  water  in  which  the  frogs  were, 
but  not  in  the  water  Occupied  by  control  frogs.  The  possible  origin 
of  this  sugar  in  the  slime  makes  it  desirable  to  repeat  this  crucial  ex- 
periment. Although  the  view  most  commonly  held  is  that  phlorhizin 
acts  specifically  and  exclusively  on  the  kidney  cells  this  has  never 
been  proved,  and  there  is  much  to  suggest  a  general  cell  effect  ex- 
hibited most  strikingly  in  the  kidney.     • 

Regarding  the  fundamental  nature  of  the  action  of  phlorhizin, 
nothing  satisfactory  has  been  evolved.  Minkowski  suggested  that 
phloretin  and  sugar  are  split  apart  in  the  kidne.y  epithelium,  and  that 
the  sugar  is  then  excreted  while  the  phloretin  is  retained  in  the  body. 
The  retained  phloretin  then  takes  up  a  new  molecule  of  glucose  from 
the  blood  to  reform  phlorhizin, — which  in  the  kidney  is  again  split, 
etc.  (vehicle  theory).  Zuntz  has  determined  with  a  given  minute  dose 
of  phlorhizin  how  much  sugar  can  be  eliminated  in  a  given  time  by  one 
kidney;  then,  figuring  what  weight  of  phlorhizin  is  in  the  kidney,  and 
how  much  sugar  comes  out  of  the  kidney,  he  reckons  how  frequently 


PHLOIiHIZIX  DIABETES  (Wi!) 

the  synthesis  and  hydrolysis  of  phlorhizin  would  have  to  occur.  He 
makes  it  26  times  per  minute,  which  he  deems  too  fast  to  be  probable, 
but  in  view  of  the  work  which  can  be  accomplished  by  traces  of  organic 
and  inorganic  carriers  (catalyzers,  enzymes),  this  criticism  is  not  con- 
vincing. 

Whatever  the  action  of  phlorhizin  may  prove  ultimately  to  be,  this 
action  finds  its  chief  or  final  expression  in  the  cells  of  the  kidney,  and 
there  leads  to  a  disturbance  of  equilibrium,  whereby  the  relative  blood 
sugar  and  urinary  sugar  concentrations  are  altered  in  favor  of  the  urine. 
The  blood  sugar  must  be  in  equilibrium  with  the  sugar  content  of  the 
various  cells,  and  this  with  the  sources  (glycogen  and  protein)  from 
which  the  sugar  comes.  The  sugar  of  the  entire  body  may  be  conceived 
of  as  a  gas  exerting  its  partial  pressure  in  every  cell  and  body  fluid, — 
here  more  dense,  there  less  so,  depending  upon  local  phj-sico-chcmical 
conditions,  but  nevertheless  everywhere  in  communication.  Piilor- 
hizin  acting  in  the  kidneys,  and  regardless  of  a  possible  action  elsewhere, 
creates  a  void  into  which  the  blood  sugar  flows,  and  into  which  second- 
arily, as  into  a  vortex,  sugar  flows  from  all  the  sources  of  the  body. 

Metabolic  Phenomena.^ — When  a  fasting  dog  is  kept  continuously 
under  the  maximum  effects  of  phlorhizin,  there  is  at  first  a  very  great 
glycosuria  while  the  urinary  nitrogen  remains  low.  The  ratio  of  the 
urinary  glucose  to  the  urinary  nitrogen  (G  :  N  ratio)  may  be  as  high 
as  10  or  15  to  1,  or  higher.  If  such  a  dog  is  killed  the  liver  is  found 
to  have  a  normal  appearance  and  to  contain  glycogen.  As  time  goes 
on  the  rate  of  glucose  excretion  falls  and  the  nitrogen  tends  to  increase, 
until  after  two  or  three  days  the  G  :  N  ratio  is  about  3.65  to  1,  as 
shown  by  Lusk.  Then  for  12  to  24  hours  it  may  remain  constant. 
It  sometimes  happens  that  the  ratio  falls  to  2.8  or  some  point  between 
3.65  arid  2.8  before  constancy  is  established.  It  then  proceeds  at  this 
lower  level  instead  of  3.65.  If  a  dog  is  killed  at  about  the  time  con- 
stancy is  attained,  or  somewhat  sooner,  the  liver  may  be  found  in  a 
state  of  fatty  infiltration  with  the  glycogen  low  but  not  absent.  In 
later  stages  the  excessive  fat  in  the  liver  again  disappears.  There  is 
then  first  a  rapid  loss  of  glucose  and  a  simultaneous  melting  away  of 
glycogen.  To  compensate  for  the  falling  out  of  the  carbohydrate 
from  the  metabolism  there  is  an  increased  breakdown  of  protein  and 
a  rapid  mobilization  of  fat,  finding  temporary  expression  in  a  fatty 
infiltration  of  the  liver.  But  as  the  fat  reserves  run  low  the  fat  de- 
posited in  the  liver  is  utihzed.  Coincident  with  the  partial  exhaustion 
of  the  carbohydrate  reserves  of  the  body  and  the  increased  fat  and 
protein  metabolism,  acetoacetic  and  (3-hydroxy  butyric  acids  begin  to 
appear  in  the  urine,  and  since  they  are  excreted  partly  in  the  form  of 
the  ammonium  salts  the  urinary  ammonia  is  also  increased.  These 
acids  arise  from  lower  fatty  acids  having  an  even  number  of  carbon 
atoms  in  the  chain,  and  from  certain  amino-acids,  whenever  the  mix- 
ture of  fatty  acids  and  glucose  actually  metabolizing  is  too  rich  in  the 
former  in  comparison  with  the  latter. 


670  DIABETES 

However,  such  animals  are  not  free  of  glycogen.  If  they  are  sub- 
jected to  some  treatment  which  has  a  strong  glycogen  mobilizing  effect 
the  glycosuria  may  be  made  to  rise  temporarilj^,  just  as  though  the 
dog  had  been  given  a  dose  of  sugar.  Thus,  exposure  to  cold  sufficient 
to  cause  shivering,  the  administration  of  epinephrine,  or  an  ether  or 
nitrous  oxide  narcosis,  injection  of  acid  (and  various  other  toxic  sub- 
stances capable  of  producing  tissue  asphyxia  and  acidosis),  all  may 
increase  the  urinary  glucose  without  increasing  the  nitrogen,  and 
thus  cause  an  increased  G  :  N  ratio.  But  if  the  exposure  to  cold  is 
long  and  intense  enough  a  time  comes  when  it  ceases  to  have  this  effect, 
and  if  epinephrine  is  given  subcutaneously  in  the  dosage  of  about  0.4 
mg.  per  kg.  of  body  weight  once  every  three  hours  there  is  for  a  time 
a  heavy  increase  of  the  glucose  output,  but  this  becomes  less  and  less 
until  after  6  or  8  doses  the  ratio  becomes  constant  again,  regardless  of 
whether  epinephrine  is  given  or  not.  In  such  dogs  neither  cold  nor 
narcosis  nor  other  toxic  effects  will  increase  the  output  of  glucose, 
and  analyses  of  the  liver  and  muscles  reveal  no  glycogen.  In  a  long 
series  of  dogs  so  treated  Sansum  and  the  writer  have  not  encountered 
ratios  above  3.2  to  1,  and  the  2.8  ratio  recurs  frequently. 

Since  the  glycogen  is  gone  and  the  dog  is  fasting,  the  sugar  which 
continues  to  appear  in  the  urine  must  have  its  origin  in  body  fat  or 
protein,  or  both. 

Sugar  from  Fat. — If  such,  a  dog  be  given  large  quantities  of  fat 
in  the  diet  no  change  occurs  in  the  G  :  N  ratio,  nor  any  increase  in 
the  glycosuria,  except  such  as  may  be  ascribed  to  the  glycerol  of  the 
fat  (Lusk).  On  the  other  hand,  propionic  acid,  according  to  Ringer, 
may  cause  a  rise  in  the  sugar  excretion  and  a  corresponding  rise  in  the 
G  :  N  ratio. "^^  From  this  it  is  concluded  that  the  fats  of  the  food  do  not 
as  a  rule  form  sugar  in  the  body,  although  sugar  formation  from  at 
least  one  lower  fatty  acid  is  possible  in  view  of  Ringer's  experiment. 

Von  Noorden  and  Falta  and  their  associates  have  regarded  sugar 
formation  from  fat  as  a  regular  normal  phenomenon,  because  in  dia- 
betes melitus  they  believe  that  high  ratios  occur  which  make  this  view 
necessary. 

Sugar  from  Protein.— If  instead  of  fat,  protein  be  given  to  the 
dog  above  mentioned,  there  occurs  an  absolute  rise  in  the  sugar  of 
the  urine  and  a  corresponding  rise  in  the  nitrogen,  bid  the  G  :  N  ratio 
remains  constant.  Following  a  meat  feeding  there  may  be  fluctuations 
of  the  ratio  during  short  periods,  but  this  statement  generally  holds 
if  the  time  of  observation  is  12  to  24  hours.  These  facts  have  led  Lusk 
to  the  conclusion  that  when  in  a  fasting,  fully  phlorhizinized  animal, 
or  one  fed  on  meat  and  fat  alone,  a  constant  G  :  N  ratio  of  3.0")  :  1 
is  seen;  this  means  that  the  glucose  and  the  nitrogen  arc  coming  from 
one  and  the  same  source,  viz.,  protein.     A  gram  of  nitrogen  corresponds 

■"  The  dogs  used  liy  Ringer  were  not  free  of  glycogen  and  possibly  the  extra 
sugar  did  not  arise  from  the  acid  given. 


THE  PANCREAS  AND  DIABETES  071 

to  6.25  grams  protein,  and  if  for  oucli  0.25  Krarus  i)r()t<'iii  iiiotabolized 
as  indicated  by  the  N  in  the  urine,  3.65  grams  glucose  are  excreted,  then 
58  per  cent,  of  the  protein  motaboiized  is  converted  into  ghicose  and 
so  excreted.  In  like  manner  the  2.S  :  1  ratio  would  indicate  a  45 
jier  cent,  conversion.  A  perccMitaKc  above  58  has  not  bo('nsat.isfactf)rily 
proved  to  occur.  If  to  the  fully  phlorhizinized  dog  a  definite  quantity 
of  glucose,  galactose,  starch  or  other  assimilable  form  of  carbohydrate 
is  given,  this  may  under  favorable  circumstances  be  excreted  quanti- 
tatively in  the  urine  as  glucose,  and  the  ratio  of  G  :  N  will  rise.  The 
sugar  which  appears  in  the  urine  under  such  circumstances  over 
and  above  that  represented  by  N  X  G  :  N  has  been  called  "extra 
sugar"  by  Lusk. 

If  all  the  carbon  contained  in  protein  were  converted  into  glucose, 
and  all  this  excreted  together  with  the  nitrogen,  the  G  :  N  ratio  would 
be  8.25:  1.  A  higher  ratio  than  this  would  necessarily  mean  that 
sugar  was  coming  from  some  source  other  than  protein,  or  that  all 
of  the  N  was  not  appearing  in  the  urine,  some  being  retained  in  the 
body.  If  the  liver  were  free  from  glycogen  and  no  carbohydrate 
were  eaten,  such  a  high  ratio  would  speak  in  favor  of  sugar  formation 
from  fat.  Falta  reports  having  seen  cases  of  diabetes  in  which  this 
occurred,  but  in  human  cases  it  is  difficult  to  be  sure  of  the  absence  of 
glycogen  and  food  carbohydrate;  moreover,  such  high  ratios,  unless 
too  long  continued,  might  imply  retention  of  nitrogen. 

Sugar  from  other  Substances. — A  large  number  of  other  substances 
when  administered  to  phlorhizinized  dogs  are  capable  of  increasing 
the  output  of  sugar.  Of  importance  in  this  connection  are  certain  of 
the  amino  acids,  viz;  glj^cine,  alanine,  aspartic  and  glutamic  acids, 
and  arginine.  Others,  such  as  leucine,  tyrosine  and  phenyl  alanine 
do  not  form  sugar  in  the  bod}^  but  increase  the  output  of  the  acetone 
substances.  The  sugar-forming  power  of  protein  is  doubtless  due  to 
its  content  of  the  former  group  of  amino  acids."*-  Lactic  a.cid  and  gly- 
cerol are  also  among  the  sugar  formers. 

The  chief  interest  in  phlorhizin  diabetes  lies  in  the  opportunities 
it  offers  of  studying  the  character  of  the  intermediate  metabolism 
minus  that  of  sugar,  and  so  of  studying  sugar  metabolism.  Another 
interest  might  be  found  were  the  physiologic  effects  of  this  glucoside 
in  animals  interpreted  with  relationship  to  its  normal  role  in  plant 
physiology. 

PANCREAS,  DIABETES  AND  DIABETES  MELITUS 

Historical. — In  1788  Cawley  reported  atrophy  and  stone  of  the 
pancreas  in  a  case  of  diabetes.  The  coincidence  of  diabetic  symp- 
toms and  lesions  of  the  pancreas  was  further  studied  by  Bright,  Lloyd 
and  Elliotson  (1833).     It  was  Bouchardat"  who  first  definitely  for- 

■•2  See  Dakin,  Jour.  Biol.  Chem..  1913  (14),  155. 

"  "De  la  Glycosurie,  etc.,"  II  edit.,  Paris,  1883.     Cited  from  Naunyn. 


672  DIABETES 

mulated  the  belief  that  pancreatic  disease  was  the  cause  of  diabetes 
melitus,  but  his  views  were  uncongenial  to  the  clinicians  of  his  time 
and  it  remained  for  von  Mering  and  Minkowski"  (1889)  to  prove 
that  complete  pancreatectomy  leads  invariably  to  the  development  of  a 
severe  diabetes.  This  applies  not  only  to  dogs  but  to  cats,  rabbits, 
pigs  (Minkowski),  tortoises,"*^  frogs;*^  eels,"*^  and  other  animals. 

Effects  of  Pancreas  Extirpation. — The  glycosuria  begins  soon  after 
the  operation  and  increases  in  intensity.  It  persists  in  spite  of  a 
non-carbohydrate  diet  long  after  the  glycogen  reservoirs  in  the  liver 
and  muscles  have  become  greatly  impoverished  (to  0.1-0.2  per  cent, 
in  the  liver),  but  like  the  human  disease,  it  usually  ceases  during  a 
fast  or  may  disappear  just  before  death. *^  The  glycosuria  may  be 
accompanied  by  an  excretion  of  the  acetone  bodies, — acetone,  aceto- 
acetic  and  j3-hydroxybutyric  acids.  In  fact,  the  metabolic  changes 
secondary  to  this  operation  closely  parallel  those  found  in  the  human 
disease,  with  certain  differences  which  perhaps  are  ascribable  to  species 
or  to  the  fact  that  in  the  experimental  diabetes  digestion  is  altered  by 
absence  of  the  pancreatic  juice,  etc.  Although  Minkowski's  work  was 
assailed  from  many  quarters,  the  following  points  have  become  firmly 
established  by  frequent  repetition.  (1)  Complete  removal  of  the 
pancreas  causes  a  true  diabetes  (as  above);  (2)  Ligation  or  oblitera- 
tion of  the  duct  (or  ducts)  of  Wirsung,  no  matter  how  scrupulously 
carried  out,  has  no  such  effect;  (3)  If  about  one-fifth  of  the  pancreas 
with  its  arterial  supply  be  separated  from  the  rest  of  the  gland,  this 
fifth  may  be  implanted  extraperitoneally  at  a  distance  from  the  origi- 
nal site.  No  diabetes  results  from  this  operation,  or  at  most  only  a 
transient  glycosuria.  Now  if  the  main  body  of  the  pancreas  be  fully 
extirpated  with  ducts,  nerves  and  bloodvessels,  still  only  a  transient 
glycosuria  or  none  at  all  develops.  At  this  stage  all  possible  damage  to 
nerves  and  external  secretion  has  been  inflicted  and  proved  incapable 
of  causing  diabetes.  (4)  In  the  course  of  weeks  the  graft  atrophies 
(Sandmeyer's  experiment),  and  then  a  persistent  glycosuria  supervenes; 
or  the  encapsulated  fragment  which  has  been  placed  in  an  accessible 
place  under  the  skin  may  be  extirpated,  in  which  case  within  a  few 
hours  a  severe  diabetes  ensues.  (5)  There  is  no  other  organ  in  the 
body  extirpation  of  which  has  any  similar  effect,  nor  (except  for  phlor- 
hizinization),  is  there  any  known  means  of  experimentally  producing 
a  true  diabetes  without  injury  to  the  pancreas.  (6)  No  toxic  sub- 
stance derived  from  the  body  of  diabetic  individuals,  man  or  animal, 
has  been  found  which  is  capable  of  causing  diabetes  in  a  second  animal. 
These  facts  lead  to  the  conclusion  {reached  by  Minkowski)  that  pancreatic 

**  Arch,  flir  cxp.  Path.  u.  Pharni.,  1889  (2G),  371;  1803  (31),  85. 

"AldchofT,  G.  Zeit.  f.  Biol.,  1891-2  (28),  293;  Velich,  Wien.  Med.  Zeitung. 
1895  (40),  502;  Marcuse  W.,  Zeit.  f.  klin.  Mod.,  1894  (2G),  225. 

■"«  CapparcUi,  Biol.  Zcntralbl.,  1893  (13),  495. 

*'  This  statement,  based  on  experimental  work,  appears  in  the  2d  (1914)  edition 
of  this  book. 


THE  PANCREAS  AND  DIABETES  G73 

tissue  provides  "a  something,"  separate  from  the  pancreatic  juice, 
(internal  secretion  of  the  pancreas),  the  lack  of  irhich  is  responsible  for 

the  sytn])toms  of  diabetes. 

Islet  Theory:  MorpholofjjiciiUy  tlie  ixincrcns  may  be  reKiinlod  a.s  Htroma, 
ducts,  acini  and  islamls  of  LanRcrhans.  It  lias  been  proposed,  notably  by  Opio" 
in  this  country,  tliat  tho  antidiabetic  internal  secretion  of  the;  pancreas  is  elabo- 
rated by  islet  cells.  This  view  hnds  support  in  the  following  facts:  (1)  In 
diabetes  melitus  the  islets  arc  frequently  found  in  a  state  of  hydropic  or  hyaline 
degeneration,  while  the  reniainiiifz;  organ  may  appear  normal.*' '  (2)  Cancer,  pan- 
creatitis and  the  experimental  injection  of  caustics  into  the  ducts  very  frecjuently 
spare  the  islets  and  fail  to  cause  diabetes.  (3)  It  is  claimed  tluit  in  pancreatic 
grafts,  such  as  described  above,  islet  cells  predominate,  while  acinus  cells  and 
ducts  disai)pear. 

Grafts  of  this  kind  consist  of  much  connective  tis.sue,  generally  more  or  less 
infiltrated  with  round  cells,  and  collections  of  epithelium.  Concerning  the  latter, 
remains  of  ducts  and  acini  are  usually  present  in  some  proportion,  andihcre  are 
also  epithelial  cell  masses  regarded  as  islets  on  mor])hological  grounds.  Differ- 
ences of  opinion  still  exist  as  to  the  relative  proportion  of  tlie  ditTerent  epithelial 
elements.  Lombroso,""  whose  exhaustive  monograph  reviews  tlie  literatute  to 
1910,  concludes  that  the  internal  function  of  the  pancreas  is  not  mono|)olized  'by 
islet  cells.  Bensley^'  developed  intra-vital  staining  methods  which,  for  the  first 
time,  made  possible  the  sure  differentiation  of  islet  cells  from  duct  or  acinus 
epithelium  without  reference  to  form  or  arrangement,  and  appears  to  ha\e  i)rovcd 
that  these  cells  are  regenerated  from  duct  epithelium.  He  also  showed  the  great 
normal  variations  in  size  and  number  of  islets  in  different  individuals  (guinea- 
pigs).  His  study  explains  certain  of  the  discrepancies  which  occur  in  the  litera- 
ture, especially  in  the  estimation  of  the  quantity  of  islet  tissue  in  j)ancreatic 
rests,  grafts,  etc.  AUen^-  has  reported  that  when  proper  sized  fragments  of 
pancreas,  in  connection  with  the  ducts,  are  left  in  situ,  and  the  remainder  of  the 
gland  is  removed,  the  subsequent  development  of  severe  diabetes  maybe  coincident 
with  disappearance  of  islet  tissue  while  acinus  cells  and  ducts  are  unaffected. 
This  operation,  according  to  Allen,  is  eminently  satisfactory  for  producing  ex- 
perimental diabetes  without  infection  and  without  loss  of  the  external  secretions. 

The  Nature  of  the  Internal  Secretion  of  the  Pancreas. — 

Direct  evidence  on  this  subject  i»  lacking.  Such  a  secretion  has  never 
been  isolated.  Even  the  experiments  made  with  the  feeding  of  fresh 
pancreas  and  with  extracts  of  the  gland  have  led  to  no  solid  advance. 
Reports  of  improvements  following  the  administration  of  any  substance 
in  diabetes  are  worthless  unless  accompanied  by  proof  of  the  constancy 
of  the  diet,  of  the  amount  of  work  perfomed,  and  of  other  factors  which 
are  known  to  influence  the  course  of  diabetes.  Some  glimmer  of  suc- 
cess appeared  to  have  attended  the  intravenous  use  of  an  extract  made 
by  Zuelzer/^  although  deleterious  by  effects  occured,  and  the  apparent 
improvement  could  have  been  due  wholly  to  retention.  According 
to  Hedon  and  Drennan,  amelioration  of  the  severity  of  pancreas 
diabetes  as  evidenced  by  a  diminution  of  glycosuria  has  followed  the 
transfusion  of  blood  from  a  healthy  animal  or  the  injection  of  fresh 
defibrinated  blood,  and  Forschbach,  working  with  a  parabiosis  (or 

*8  "Diseases  of  the  Pancreas,"  Lippincott  &  Co.,  1910. 
"  See  Homans.  Jour.  Med.  Res.,  1914  (30),  49. 
soErgeb.  der  Phvsiol.,  1910  (10),  1. 
"  Am.  Jour,  of  Anat.,  1911  (12),  297. 
^2  Glycosuria  and  Diabetes,  Boston.  1913. 
"  Zeit.  f.  exp.  Path.,  1908-9  (5),  307. 
43 


674  DIABETES 

two  animals  so  joined  by  operative  means  that  permanent  inter- 
mingling of  their  blood  occurs)  performed  pancreatectomy  in  one  of 
the  animals  without  producing  diabetes  in  either;  from  which  it  might 
seem  that  the  internal  secretion  was  carried  by  the  blood.  In  harmony 
with  these  results  were  the  investigations  of  Knowlton  and  Starling,^'* 
who  found  that  an  isolated  beating  heart  taken  from  a  depancreatized 
animal  (cat)  was  capable  of  removing  less  sugar  from  the  blood  used 
as  a  perfusion  medium  than  are  hearts  of  normal  animals,  but  these 
latter  experiments  have  not  been  confirmed  and  are  subject  to  criticism. 
In  most  of  the  transfusion  experiments  reported  the  standardization 
of  the  metabolism  prior  to  giving  the  fresh  blood  has  not  been  such 
as  to  make  the  results  certain.  Carlson  and  Drennan  found  that 
pancreatectomy  in  a  pregnant  animal  near  term  might  fail  to  cause 
diabetes,  but  that  diabetes  developed  at  once  following  delivery.  This 
could  be  explained  on  the  basis  that  an  internal  secretion  passed  from 
fetus  to  mother,  or  that  sugar  failing  of  utilization  in  the  mother  was 
utilized  by  the  fetuses.  Kramer  and  Murlin  failed  to  note  any  increase 
of  the  respiratory  quotient  in  depancreatized  dogs  following  blood 
transfusion,  and  Sansum  and  Woodyatt  saw  no  improvement  following 
transfusion  in  a  human  case."  Recently  Kleiner^^"  has  reported  a  di- 
minution of  the  total  blood  sugar  in  dogs  following  infusion  of  pan- 
creas emulsion,  and  this  work  revives  the  interest  in  a  problem  of  great 
importance. 

Symptoms.^ — ^In  the  absence  of  extracts  which  contain  the  active 
principle  in  measurable  quantity,  the  attention  must  be  turned  to  a 
more  detailed  study  of  the  effects  which  follow  its  lack.  Now  it  is 
well  known  that  in  diabetes  melitus  there  are  all  grades  of  severity. 
What  follows  has  reference  only  to  the  severest  cases — those  which  may 
be  called  "complete  diabetes."  In  the  severest  cases  of  diabetes,  gly- 
cosuria persists  even  when  the  individual  subsists  on  a  fat-protein 
diet,  and  after  the  glycogen  in  the  body  has  been  reduced  to  a  mere  trace. 
When  this  stage  has  been  reached,  and  provided  no  carbohydrate  food 
is  eaten,  it  is  found  that  the  total  glucose  in  the  urine  bears  from  day 
to  day  a  constant  ratio  to  the  total  nitrogen  in  the  urine  as  already 
described  for  phlorhizin  diabetes.  This  "G  :  N  ratio"  is  not  always 
the  same.  In  depancreatized  dogs  nourished  solely  on  fat  and  pro- 
tein, it  is  often  found,  as  Minkowski  first  recognized,  at  2.8  :  1,  and 
in  human  diabetes  the  same  value  for  G  :  N  is  sometimes  seen.  But, 
as  in  phlorhizinized  dogs,  higher  ratios  may  occur  in  the  human  disease. 

If  to  such  a  case  of  diabetes  as  this  we  give  b}--  mouth  40  grams  of 
glucose  there  may  appear  in  the  urine  close  to  40  grams  of  extra  sugar. 
Plainly  such  extra  sugar  has  escaped  utilization  of  any  kind.  It  can- 
not have  been  oxidized  or  converted  into  fat,  since  these  processes  are 

"  Jour,  of  Physiol.,  1913  (45),  146. 

''^  Jour.  Amor.  Med.  Assoc,  1914  (02),  99(5  for  lit.  references. 

""Jour.  Biol.  Cheiii.,  1919  (40),  153. 


TIIEOUY  OF  DIMiKTES,  675 

irrcvcrsiblo,  although  it  mij^ht  luivc;  (\\i.st(Ml  iiujiucnturily  in  tlic  Ixjdy 
as  glycogen  or  other  isomer  of  ghicosc.  What  j)haHe  in  tin;  utihzation 
of  this  glucose  is  primarily  disturbed  is  another  fjuestion.  To  say 
that  40  grams  of  ingested  glucose  causes  the  appearance  of  40  grams 
of  extra  sugar  in  the  urine  does  not  prove  that  tlu;  diabetic  body  is 
inherently  incapable  of  using  any  sugar  or  every  carbohydrate.  It 
might  still  be  capable  of  using  a  two,  three,  or  four  carbon  atom  sugar, 
some  other  member  of  the  group  of  32  hexoses,  or,  as  some  have  it  (von 
Noorden),  sugar  which  has  first  been  built  up  into  glycogen,  etc., 
provided  these  substances  could  be  kept  from  undergoing  transfor- 
mations into  the  non-utilizable  glucose.  As  a  rule,  however,  when 
other  sugars  are  fed  to  complete  diabetics,  they  are  transformed  into 
glucose  and  appear  as  such  in  the  urine.  This  phenomenon  has  much 
of  significance  for  the  general  theory  of  sugar  metabolism  and  is  an 
indication  of  the  nature  of  the  primary  disturbance  in  diabetes,  as  will 
now  be  shown. 

Theory  of  Diabetes. — What  sort  of  a  chemical  process  is  involved 
when  levulose,  for  example,  is  converted  in  the  body  into  glucose? 
As  already  stated  in  the  chemical  introduction,  the  reciprocal  trans- 
formations of  hexoses  one  into  another  in  the  alkaline  solution  in 
vitro  depend  upon  a  preliminary  ionization  of  the  sugars  followed  by  salt 
formation,  the  salts  then  undergoing  dissociation  which,  according 
to  Mathews  and  Michaelis,  is  still  purely  electrolytic  with  rearrange- 
ments of  the  anion;  but  which,  according  to  Nef,  is  a  non-electrolytic 
dissociation  of  the  type  which  he  calls  methylene  dissociation.  Some 
form  of  dissociation  must  he  a  prelude  also  to  these  transformations  in 
the  body.  This  view  is  logically  just  as  necessary  as  it  has  been  found 
to  be  for  the  organic  chemist,  and,  it  may  be  added,  that  for  the  oxida- 
tion of  sugars  as  well  as  for  their  polymerization  a  preliminarj'  dissoci- 
ation is  essential.  Now  since  the  diabetic  body  can  transpose  other 
sugars  into  glucose,  it  must  be  able  at  least  to  dissociate  the  former 
sugars  deeply  enough  for  this  process.  These  transpositions  are  ac- 
complished chiefly  in  the  portal  system  and  perhaps  in  other  places 
too,  but  certainly  levulose  and  many  other  substances  can  be  made  in 
the  liver  into  glycogen,  whose  hydrolysis  then  yields  glucose. 

The  degree  or  character  of  the  dissociation  necessary  for  reciprocal 
transformations  differs  from  that  which  is  a  necessary  prelude  to  de- 
structive reactions  such  as  oxidation.  A  very  weak  alkali  suffices 
in  vitro  for  the  former,  while  for  the  latter  it  is  necessary  to  use  a 
somewhat  stronger  alkali  concentration.^^  The  diabetic  body  there- 
fore behaves  as  though  it  were  weakened  with  respect  to  the  alkali 
concentrations  which  it  can  bring  to  bear  on  sugars. 

As  far  back  as  1871,  Schultzen  suggested  that  the  error  in  diabetes 
might  be  found  in  the  disability  of  the  body  to  dissociate  the  glucose 

"See  Woodyatt,  Jour.  Biol.  Cheni.,  1915  (20),  129. 


676  DIABETES 

molecule  into  two  3-carbon  substances. ^^  Baumgarten^^  also  sup- 
ported the  idea  of  a  "fermentative  splitting"  which  precedes  oxida- 
dation,  because  he  found  a  greater  percentage  utilization  of  certain 
substances  closely  allied  to  glucose  (such  as  gluconic  acid,  saccharic 
acid,  mucic  acid,  etc.),  than  of  glucose  itself;  whereas  gluconic  acid 
and  glucose,  for  example,  differ  only  in  that  the  sugar  has  an  aldehyde 
group  where  the  acid  has  carboxyl.  Similar  general  ideas  have  been 
expressed  from  time  to  time  by  others.  The  present  writer  has  urged 
in  place  of  the  vaguer  terms,  the  adoption  of  chemical  "dissociation" 
in  the  sense  which  is  rapidly  finding  favor  in  the  field  of  pure  organic 
chemistry,  notably  for  the  explanation  of  the  behavior  of  aldehj^des, 
ketones  and  alcohols. ^^  There  can  be  no  doubt  that  the  dissociation 
of  glucose  in  the  body  is  a  normal  occurrence.  This  is  directl}'  and 
conclusively  shown  whenever  muscles  make  lactic  acid  (CsHeOs) 
out  of  glucose  (C6H12O6),  since  in  this  process  no  chemical  phenomenon 
is  involved  save  cleavage  of  the  hexose  and  intramolecular  rearrange- 
ment. The  polymerization  of  sugar  into  glycogen  might  be  similarly 
interpreted.  Direct  proof  of  a  failure  of  glucose  dissociation  in  dia- 
betes has  not  yet  been  brought,  although  its  absence  would  explain 
all  the  metabolic  phenomena  more  directly  and  simply  than  anj''  other 
single  physiologic  error  which  has  been  hypothecated.  It  is,  moreover, 
a  tangible  chemical  conception,  whereas  to  say  that  the  bodj'  loses  its 
power  .to  oxidize  sugar  or  to  "fix"  it  as  glycogen  is  merel}"  to  name 
effects  in  physiologic  terms.     (Cf.  Naunyn's  diszoamylie.) 

It  might  be  assumed  that  all  sugars  upon  entering  certain  phases  of 
the  cells  (phases  especially  well  represented  in  liver  cells),  meet  con- 
ditions which  are  equivalent  to  those  met  in  a  weakly  alkaline  solution, 
favoring  reciprocal  transformations,  and,  as  A.  P.  Mathews  points 
out,  polymerization;  but  not  conditions  conducive  to  the  deeper  de- 
structive reactions.  That  is,  especially  in  the  liver,  there  may  be  the 
equivalent  of  dilute  alkali  for  all  sugars.  Glucose,  being  the  least 
dissociable,  represents  the  form  into  which  all  other  sugars  tend  to 
accumulate.  But  in  the  normal  body  a  special  glucolytic  enzyme 
(alkali  carrier  or  intensifier?)  destroys  glucose  selectively.  All 
other  sugars  must  become  glucose  before  destruction.  In  diabetes 
the  enzyme  necessary  for  the  deep  dissociation  of  glucose  is  lacking  or 
inactive.  The  recent  studies  of  Murlin,  Kramer,  Sweet  and  Karver, 
show  that  alkali  administration  (NaoCOa)  ma}'  increase  glucose  utili- 
zation, especially  when  introduced  into  the  duodenum  whore  it  may 
neutralize  acid  entering  the  bowel  from  the  stomach  and  thus  spare 
the  liver  and  pancreas  from  the  effec^ts  of  absorbed  acid,  l^ndorhill's 
experiments'^"  with  bicarbonate  feeding  in  diabetes  confirm  liiese 
observations. 

"  Glyceric  aldehyde  and  fjlyccrol,  accord iiiji;  to  Schultzen. 
'»  Zcit.  f.  exp.  Path.  u.  IMianii.,  I'.K)')  (2),  oii. 

'■'•' Cf.  Nef,  lor.  cU.,  and  Stieglitii,  Ciualilative  Chemical  .Vnalvsis,  New  York, 
1912,  I,  PI).  28<)-2«)2. 

«» Jour.  Ainer.  Med.  Assoc,  1917  (68),  497. 


THEORY  OF  DIMiErKS  077 

One  difference  between  diabetes  melitus  and  plilorliizin  diabetes  is 
that  in  the  former  the  glycosuria  is  due  to  hyperKlyceniia.  the  .sugar 
loss  being  an  overflow  like  water  escai)ing  from  an  overfdle*!  tank; 
whereas  in  phlorhizin  poisoning  there  is  apparently  an  hy|)(.glycemia 
—the  loss  resulting  in  this  case,  to  carry  out  the  simile,  from  a  leak  in 
the  bottom  of  the  tank  which  keeps  the  water  at  a  lower  level.  But  t  he 
results  are  the  same.  ]\Ioreover,  if  in  diabetes  melitus  we  could  meas- 
ure only  the  chemically  active  or  dissociated  blood  sugar,  it  is  possible 
we  should  again  find  for  this  kind  of  sugar  an  liyi)oglycemia  compara- 
ble to  that  of  phlorhizin  diabetes.  This  conception  coincides  with  the 
doctrine  that  in  diabetes  melitus  there  is  a  primary  nnderconsumption 
of  sugar  as  opposed  to  the  idea  of  a  primary  overproduction. 

Overproduction  vs.  Underconsximption. — The  chief  expf)nents  of 
overproduction  have  been  the  followers  of  Kraus,  and  of  von 
Noorden  in  whose  books  "Die  Zuckerkrankheit"  and  "New  Aspects 
of  Diabetes"  will  be  found  the  arguments  favoring  this  idea.  A 
translation  of  Minkowski's  criticism  of  the  latter  has  been  made  by 
Lusk."  In  this  place  it  may  be  briefl\'  recalled  that  the  chief  argu- 
ments favoring  underconsumption  in  addition  to  what  has  already 
been  said  are  the  followng:  (1)  The  respiratory  quotient  in  diabetes 
is  frequently  found  to  be  low,  and  w^hen  carbohydrate  food  is  admin- 
istered this  quotient  rises  but  little,  whereas  in  health  the  administra- 
tion of  carbohydrate  food  results  in  a  greater  rise.''-  (2)  The  acetone 
bodies  (acetone,  aceto-acetic  acid  and  beta-hydroxybutyric  acid) 
appear  in  the  urine  when  for  any  reason  the  quantity  of  sugar  burning 
in  the  body  falls  below  a  certain  minimum,  as  in  starvation,  or  when  a 
person  accustomed  to  a  mixed  diet  is  suddenh'  switched  to  a  full  calory 
diet  composed  exclusively  of  fat,  or  of  fat  and  carbohydrates,  with 
the  carbohydrate  calories  representing  less  than  10  percent,  and  the 
fat  calories  more  than  90  per  cent,  of  the  total  (Zeller*'\).  In  these 
cases  the  restoration  of  sugar  to  the  diet  abruptly  and  permanently 
stops  the  output  of  acetone  bodies.  But  in  severe  diabetes  the  excre- 
tion of  acetone  bodies  is  less  affected  by  the  giving  of  sugar.  Follow- 
ing single  large  doses  there  may  indeed  be  a  temporary  fall  in  the 
acidosis,  but  this  is  never  permanently  attainable.  One  interpreta- 
tion made  of  these  facts  is  as  follows.  In  diabetes  there  is  an  acetone 
body  output  because  sugar,  although  brought  to  the  cells,  fails  to  take 
part  in  certain  chemical  reactions  which  normally  occur  between 
sugars  and  certain  of  the  breakdown  products  of  butyric  acid  and 
which  normally  prevent  the  diabetic  acidosis.  Hence  the  bringing  of 
more  sugar  has  little  effect.     And  here  again  one  might  suggest  that 

"  Medical  Record,  Feb.  1,  1913. 

^-  For  the  literature  of  respiration  studies  in  diabetes  see  Joslin,  Treatment  of 
Diabetes  ]\Ielitus,  New  York,  1916;  Du  Bois,  Harvey  Society  Lectures,  191(i;  and 
"Studies  from  the  Department  of  Physiology  of  Cornell  University,  1915  ct  seq.; 
published  in  the  Archives  of  Internal  Medicine  and  reprinted  as  Bulletins. 

"  Arch.  f.  Physiol.,  1914,  p.  213. 


678  DIABETES 

in  diabetes  glucose  fails  to  interact  with  the  products  mentioned 
because  the  glucose  is  not  sufficiently  dissociated.  Another  inter- 
pretation has  been  to  the  effect  that  the  sugar  simply  causes  a  com- 
pensatory decrease  of  the  fat  metabolism,  i.  e.,  spare  fat,  thereby 
decreasing  the  formation  of  the  acidosis  bodies.  The  mechanism  of 
the  process  is  in  any  case  still  a  theme  for  research. 

There  are  numerous  other  theories  of  diabetes,  for  the  presentation 
of  which  the  reader  is  referred  to  the  larger  works.  Lepine  has  long 
stood  for  the  view  that  the  pancreas  secretes  a  glycolytic  oxidizing 
ferment.  Naunyn's  theory  pays  particular  regard  to  the  ability  of 
the  body  to  "fix"  glycogen,  while  glycogen  formation  is  held  to  be 
a  necessary  preliminary  step  in  the  utilization  of  sugar.  The  failure 
to  fix  glycogen  he  calls  "diszoamylie,"  and  the  other  metabolic  dis- 
turbances he  regards  as  sequences.  The  complex  development  of 
this  same  general  idea  by  von  Noorden,  with  the  added  element  of 
primary  sugar  overproduction,  has  already  been  alluded  to.  Pavy 
saw  in  the  diabetic  a  failure  to  assimilate  sugar;  that  is,  a  failure  of 
the  body  to  incorporate  sugar  in  a  colloidal  combination  which  would 
at  once  permit  of  its  transportation  to  the  points  of  utilization,  and 
prevent  its  premature  excretion.  The  assimilation  he  held  occurred 
in  the  villi  of  the  intestines,  and  the  lymphocytes  he  regarded  as  the 
morphologic  elements  which  carry  the  sugar.  Cohnheim's  theory 
that  the  muscle  formed  glycolytic  enzymes,  for  which  the  pancreas 
supplies  an  essential  activator,  is  without  any  substantial  experimental 
support  at  the  present  writing.  Allen  proposed  that  the  pancreas 
supplies  an  "amboceptor"  which  is  essential  for  the  proper  colloidal 
blood  sugar  combination.  For  a  thorough  discussion  of  the  basal 
metabolism  in  diabetes  melitus  and  its  variations  during  changes  of 
diet,  etc.,  the  reader  is  referred  to  the  studies  of  Benedict,  Lusk,  Du- 
Bois,  Allen  and  others,  references  to  which  are  given  in  Allen's  mono- 
graph. 

Bronzed  diabetes,  the  name  given  to  that  form  of  hemochromatosis 
in  which,  along  with  the  hepatic  cirrhosis,  there  is  an  associated  fibrosis 
of  the  pancreas,  and,  as  a  result  of  this,  the  symptoms  of  pancreatic 
diabetes,  will  be  found  discussed  under  the  heading"  hemochromatosis, " 
chapter  xviii. 

Diabetic  coma  is  discussed  under  "acid  intoxication,"  chapterjxx. 

Lipemia,  which  is  observed  frequently  and  most  severely  in  diabetes, 
Ls  discussed  in  chapter  xvi. 

Glycogen  in  pathological  processes  is  discussed  in  chapter  xvi. 


INDEX 


Note. — The  numbers  printed  in  boldface  type  refer  to  pages  upon  which  the  topic 

i^  specificall}-  discussed. 


Abderhalden  reaction,   204-205,    305 

Abrin,  138-140 

Abrus  precatorius,  138 

Absorption  of  lymph,  338 

Acanthosis  nigricans,  474 

Acetanihd,  486 

Acetic  acid,  244,  592 

Aceto-acetic  acid,  558-563 

Acetone,  208,  558-563,  588,  589 

Aceto-nitrile,  599 

Acetonuria,  555-569 

in  pregnancy,  568 
Acetvl-choline,  593 
Acidi!  acetic,  244,  592 
•  aceto-acetic,  558-563 

agglutination,  183 

arachidic,  521 

benzoic,  583 

bile,  244 

/3-oxybutyric,  558-563,  569 

butyric,  134,  559,  565,  592 

carbolic,  367 

chondroitin-sulphuric,  422,  431,  465, 
517 

diacetic,  558-563 

ellagic,  467 

ethereal  sulphuric,  580 

fastness,  106 

fattv,  134,  208,  226,  230,  242,  270, 
272,  274,  325,  387,  403,  589,  592 

formic,  153,  351 

gentisic,  588 

glucothionic,  271 

glycothionic,  427 

glycuronic,  239,  283,  512,  580,  583, 
666 
as  protective  substance,  243 

guanylic,  630 

helvellic,  222 

hippuric,  243,  368 

homogentisic,  477,  586-589 

hydroxystearic,  413 

indole-acetic,  579,  583 

intoxication,  555-569 

isocetinic,  106 

kynurenic,  580 

lactic,  208,  251,  272,  394,  401,  501, 
544,  552,  564,  592,  653.  See  also 
Diabetes.  « 

lauric,  106 

Htho  felUc,  467 

6' 


Acid,  malic,  248 

monobromacetic,  393 

mucoitin-sulphuric,  431 

myristic,  521 

myristinic,  106 

nicotinic,  282 

nucleic,  111,  121,  162,  192,  236,  369, 
502,  629  631 

oleic,  133,  223,  414,  545 

osmic,  216,  403 

oxalic,  381,  462,  592,  633 

oxymandelic,  551 

oxy-proteic,  511 

para-oxyphenyl  acetic,  579,  583 

para-oxyphenyl-propionic,    579,    583 

phenylacetic,  580,  583 

picric,  550 

p-oxyphenyl-lactic,  551 

propionic,  579 

proteic,  307 

quillajic,  143 

sarcolactic,  542,  551,  564,  569 

sUicic,  208,  218 

skatole  acetic,  579 

succinic,  132 

sulphuric,  580 

as  defense  against  poisons,  242 

uric,  311,  357,  466,  468,  494,  510,  542, 
623,  62&-641 

uroleucic,  587 

valerianic,  134 
Acidosis,  65,  292,  205,  392,  416,  448, 
535,  538,  546,  555-569 

in  infancy,  566 

in  nephritis,  565 

starvation,  285 
Acromegaly,  623 
Actinomyces,  107,  112 
Actinosphaerium,  379 
Acute  yellow  atrophy  of  liver,  95,  647- 

555,  577 
Addison's  disease,  620-621 

pigment  of,  475 
Adenine,  282,  641 
Adipocere,  402,  412-415 
Adipose  fluids,  363 
Adiposis  dolorosa,  518 
Adrenals,  283,  475,  615-621 

lipoids  of,  616 
Adrenalin.     See  Epinephrine. 
Aethalium  septicum,  249 


■9 


680 


INDEX 


Agaricus  campestris,  141 
Age,  colloidal  changes,  371 
Agglutination  bj''  acids,  183 

of  corpuscles,  218-219 

reaction,  178-183 
Agglutinins,  178-183,  295,  309 
Agglutinogen,  178 
Agglutinoids,  180 
Aggressins,  125 
Air  embolism,  326 
Albinism,  471 
Albumin,  20,  184,  209,  351 
Albuminuria,  535 
Albumose,  45,  64,  87,  91,  94,  98,  135, 

167,  271,  274,  310,  362,  390,  445,  535, 

575-578.     See  also  Proteose. 
Albumosuria,  271,  501,  577 

myelopathic,  525-529 
Alcohol,  242 

ascaryl,  135 

cetyl,  521 

eikosyl,  521 

ethyl,  80 

oxidase,  66 

tolerance  to,  238 
Alcoholic  fermentation,  53 
Alcoholism,  415 
Aldehvdase,  66,  68,  95,  553 
Aldehydes,  134,  375 
Aldose,  653 
Alkalosis,  533,  566 
Alkaptonuria,  477,  586-589 
Allantoin,  357,  631,  623 
Allergy,  191-203 
AUoxuric  bodies,  627 
Alphanaphthol,  69 
Altmann's  granules,  93 
Amanita  hemolysin,  222 

muscaria,  140 

phalloides,  140,  161 

toxin,  141,  161 
Amblyopia,  611 
Amboceptor,  207-209,  214,  229 

hemolytic,  215-216 
Ambrosia,  203 
Amebffi,  75 

artificial,  Rhumbler's,  262 

coli,  129 
Ameboid  motion,  248-267 

artifitiial  imitations  of,  260-263 
Amibodiastase,  256 
Amines,  87.     See  also  Pressor  bases. 
Amino-acids,  19,  77,  95,  102,  112,  171, 

192,    274,    279,    311,    391,    500,    537, 

542,  563,  589,  671.     See  also  Acute 

yellow  atro-phy. 
Amino-ethyl  alcohol,  23 
Ammonia,  533.     See  also  Acidosis. 
Ammonio-magnesium  phosphate,  462 
Ammonium  carbamate,  533 

salts,  212 
Amoeba.     See  Ameh(p. 
Amylase,   61,   71,   73,-74,90,   98,    110, 

387,  507 


Amyloid,  421-427,  428,  465 
Amvlopsin.  56 
A-naphthol.  243 
Anaphylactin,  201 
Anaphvlactogens,  191 
Anaphvlatoxin,  194-201,  251 
Anaphylaxis,    131,    135,    191-203,   573, 
586 

metabolism  in,  198 

passive,  201 
Ancistrodon  contortrix,  142 

piscivorus,  142 
Anemia,  65,  220,  225,  301-312,  317,  478, 
595 

bothriocephalus,  133 

local,  371 

pernicious,  227,  305-308 

secondary,  301-303 
Anesthesia,  567 
Angioneurotic  edema,  352 
Aniline,  213,  486,  498 
Animal  parasites,  128-137 
chemistry  of,  128-137 
glycogen  in,  436 
Ankylostoma,  136 
Anthracene,  499 
Anthracidal  serum,  209 
Anthracosis,  469 
Anthrax  bacillus,  105,  125,  268 
Antiamylase,  74 
Anticatalase,  64 
Anticoagulin,  318 
Anticomplement,  208,  234 
Antiemulsin,  61 
Anti-endotoxins,  125 
Anti-enzymes,  57-62,  269 

autolvtic,  83 
Antiferments,  57-62,  200 
Antigens,  160-165,  229 

lipoids  as,  162-163 

non-protein,  161-165 

simple  chemical,  163 
Antihemolysin,  161,  217,  226 
Antimony,  241 
Anti-oxidases,  59 
Antipapain,  61 
Antipepsin,  59 
Antiplatelet  .serum,  234,  300 
Antipneumin,  63 

Antiprotease,  61,  113,  205,  269,!361 
Antirennin,  59,  149 
Antiricin,  162,  221 
Antiserum,  cholera,  207 

parathyroid,  235 
Antithrombin,  295,  298,  316,  321 
Antithyroid  serum,  235,  612 
Antitoxin,  168.  172-177 

chemical  nature  of,  175-177 
Antitrypsin,  57-62,  134,  170,  198,  379, 

390,  543,  603 
Ants,  154 

Aorta,  cholesterol  in,  419 
.Vphrodite  aculeata,  164 
Aporrhegma,  118 


INDEX 


681 


Arabinose,  650 

Arachidic  acid,  521 

Arachnolysin,  152 

Areas  senilis,  419 

Arginaso,  78 

Arginine,  20,  91,  95,  532,  553,  590 

Arsenic,  81,  240,  547 

defense  against,  240 

eaters,  237 
Arseniiiretted  hydrogen,  213 
Arterial    degeneration    from    epineph- 
rine, 619 
Arteriosclerosis,  592,  594,  617,  619 
Artificial  ameba?,  Rhunibler's,  262 
Ascaris,  134 

lunibricoides,  129 

mcgalocephala,  134 
Ascaryl  alcohol,  135 
Ascites  adiposus,  364 
Asiatic  cholera,  566 
Askaron,  135 
Asphyxia,  83 
Atrophic  cirrhosis,  566 
Atrophy,  86,  395-396 

acute  yellow,  of  liver,  95,  547-555,  577 

brown,  396,  478,  487 

serous,  of  fat,  342,  395 
Atropine,  197 

Auto-agghitination,  219,  228 
Autocytotoxins,  235 
Autodigestion,  76.     See  also  Autolysis. 
Autohemolysin,  227 
Avitointoxication,  530-596 

gastro-intestinal,  574-586 
Autolysis,  71,  75-100,  177,  195,  271, 
317,  327,  369,  378,  391,  395,  396, 
397,  398,  408,  502,  544,  596,  598, 
601,  617.  See  also  Acute  yellow 
atrophy 

histology  of,  92,  94,  97 

in  leukemia,  98 

in  necrotic  areas,  92-94 

in  pneumonia,  90-92 

in  postmortem  changes,  96 

in  relation  to  infection,  97 

in  tumors,  98-100,  505 

influence  of  chemicals  on,  80 

of  bacteria,  84,  113-115 

of  fetus,  93,  97 

of  liver,  93,  95 

of  nervous  tissue,  93 

relation  of,  to  metabolism,  81 

uterine,  86 
Autolytic  antienzymes,  83 
Auto-opsonin,  228 
Auxanographic  method,  109 
Auxetics,  499 

Bacillus    aerogenes    capsulatus,    308, 

392 
anthrax,  105,  125,  268 
botulinus,  163 
coH  communis,  89,  111,  126,  583,  585, 

589 


Hacilliis.  diphtheria,   104,   10."),   10«1,   125 

emphyscMiatoHus,  4S2,  4H(» 

nu'gathoriurn,  220 

meaentericus,  104 

prodigiosuH,  1 10 

proteus.  111,  112 

pyocyancus,  110,  115,  126,  214,  219 

radicicola,  105 

subtilis,  107,  110 

tubercle,   65,   72,   94.    10."),    110,    12."), 
126,  320,  3S3,  3X4 
fats  of,  105-107 

typhosus,  12(),  178,  183,  219,  282 

Welchii,  220 

xylinum,  105 
Bacteria,  autolysis  of.  si,  113  115 

chemistry  of,"  101   125 

chemotaxis  of,  102 

hemolysis  by,  219  220 

oxidizing  enzymes  of,  112 

plasmolysis  of,  101 

plasmoptysis  of,  101 

poisonous  proteins,  125   126 
Bacterial  carbohydrates,  105 

enzymes,  109-115 

immunity  against,  112 

fats,  105-107 

pignumts,  126-127 

toxins.  120-125 
Bactericidal  power  of  blood,  314 

substances,  87,  97,  114 
of  leucocytes,  259 
Bacteriolysis,  206-210 
Barium,  381 
Basal  metabolism,  302,  602,  607,  623, 

638 
Basophile  leucocytes,  69 
Bee  poison,  153 

Bence-Jones  protein,  310,  626-529 
Benzoic  acid,  583 
Beriberi,  283 

Beta-iminazolyl-ethylamine,  584 
Beta-oxvbutvric  acid,  568-663,  .')69 
Betaine,"^  118,  119 
Bezoar  stones,  467 
Bile,  122 

acids,  244 

pigments,   297,   306,   453,  483.   488- 
496.     See  also  Gall-stones. 

salts,  214,  319,  322,  389,  453,  492- 
498 
Bilharzia,  129 
Biliary  calculi.  463-468 
Bilifuscin,  454 
Bilihumin,  454,  45S 
Bilirubin,  136,  296,  362,  375.  4.54,  481, 

483,  488-496 
Biliverdin,  454,  489 
/3-iminazolvlethvlamine,  196,  584 
Black  flies",  1.54' 
Blackwater  fever,  226 
Blastomvces,  266 
BUsters,"573 

fluid,  362 


682 


INDEX 


Blood,  bactericidal  power  of,  314 

coagulation  of,  146,  201,  299,  493 

composition  of,  290-293 

effect  of  carbon  dioxide  on,  314 

lakingof,  211 

menstrual,  321 

pigments,  273,  481-488 

plasma,  291 

platelets,  300 

pressure,  341 

reaction  of,  292.     See  also  Acidosis. 

regeneration  of,  302 

sugar  in,  649-652 

viscosity  of,  293,  315 
Bone-marrow,  85,  185 
Bordet-Gengou  reaction,  229 
Bothriocephalus,  307 

anemia,  133 
Botulinus  toxin,  122 
/3-oxybutyric  acid,  558-563,  569 
Brilliant  green,  215 
Bromin,  600,  601 
Bronchiectasis,  273 

fetal,  363 
Bronchitis,  273 
Bronzed     diabetes,     488.      See      also 

Hemochromatosis. 
Brown  atrophy,  396,  478,  487 
Brack's  reaction,  232 
Bufagin,  155 
Bufo  agua,  155 
Bufonin,  154 
Bufotalin,  154 
Burns,  362,  377 

poisons  of,  571-573 
Buthus,  150 
Butter  cysts,  522 
Butyrase,  71,  72 
Butyric  acid,  134,  559,  565,  592 


Cachexia,   60,  65,   73,  273,   302,   356, 
567,  568 
in  cancer,  510-513 
Cadaverine,  118,  589,  591 
Caffeine,  241,  627,  635 
Calcification,  385,  439-447 
iron  in,  440 
metastatic,  442,  525 
phosphoric  acid  in,  446 
Calcium,  189,  197,  244,  256,  292,  299, 
322,  360,  364,  381,  387,  394,  492, 
504,  512,  545,  613 
carbonate  calculi,  462 
gout,  443 

in  blood  clotting,  318 
metabolism.     See    Osteomalacia    and 

Rickels. 
oxalate,  454,  466 

calculi,  461 

soaps,  445,  518 

Clacospherites,  441 

Calculus,  Bezoar,  467 

biliary,  453-468 


Calculus,  calcium  carbonate,  462 
oxalate,  461 

cholesterol,  463 

cystine,  463 

fibrin,  463 

fusible,  462 

indigo,  463 

lung,  468 

pancreatic,  465 

phosphate,  462 

salivary,  466 

urate,  461 

uric  acid,  460 

urinary,  459-464  • 

urostealith,  463 

xanthine,  463 
Cammidge  reaction,  390 
Cancer,  60,  65,  73,  89,  99,  190,  227,  356 
441,  525,  577 

cachexia  in,  510-513 

colloid,  523 

gastric,  57 

hemolysis  in,  509 

immunity  reactions  in,  514-516 

metaboUsm  in,  510 

sulphur  metabolism  in,  511 

thyroid,  508 
Cantharidin,  164 
Capillaries,  permeabihty  of,  333 

walls,  permeabilitj'  of,  342 
Carbamate  ammonium,  533 
Carbohydrate  metabolism,  642-678 
Carbohydrates,  23 

bacterial,  105 

fermentation  of,  592 
Carbolic  acid,  367 
gangrene,  380 
Carbon  dioxide,  208 

effect  on  blood,  314 
Carcinoma.  See  Cancer. 
Cardiac  disease,  65 

edema,  348 
Carotid  gland,  625 
Carotin,  479 
Carotinemia,  479 
Caseation,  94,  384-386 
Casein,   160,    161,    166,   169,    192,    197, 

280,  423 
Castration  granulomas,  278,  519 
Catalase,  54,  61,  63-65,  68,  71,  112,  272, 

283,  506,  554,  603 
Catalysis,  51 
Cataphorcsis,  35 
Cataract,  375 
Cell,  chemistry  and  physics  of,  17-47 

chromophile,  480 

death,  physico-chemical  changes,  370 

division,  277 

giant,  69 

morganic  substances  of,  24 

life,  30 

lymphoid,  71 

mast,  42,  45,  310 

mechanical  injury  of,  381 


INDEX 


683 


Cell,  physical  chemistry  of,  24-39 
plant,"  28,  04,  383 
plasma,  G9 

structure  of,  39-47 

tissue,  cytolysis  of,  233-236 

wall,  45-47  ' 
Cellulose,  105 
Cement  substance,  294 
Centipedes,  153 
Cephalin,  23,  317 
Cephalopods,  158,  585 
Cerebral  hemorrhage,  297 
Cerebrosides,  504 
Cerebrospinal  fluid,  359-362 
Cerolipoids,  106 
Cestodes,  131-134 
Cetyl  alcohol,  521 
Chalicosis,  469 
Charcot's  crystals,  311 
Chemoreceptors,  237 
Chemotaxis,  248-267,  314 

of  bacteria,  102 

theories  of,  259 
Chemotropism,  249 
Chitin,  105,  128,  132 
Chitosamine,  431,  520 
Chlorides,  retention  of,  350 
Chloroform,  72,  73,  80,  95 

necrosis,  321 

poisoning,  548,  567 
Chloroma,  480 
Chlorophyll,  126,  485 
Chlorosis,  303-305 
Cholemia,  494 
Choleprasin,  489 
Cholera,  590 

antiserum,  207 

Asiatic,  566 

vibrios,  103 
Cholesteatomas,  522 
Cholesteatomatous  tumors,  419 
Cholesterase,  73 

Cholesterol.  23,  29,  45,  79,  106,  132, 
155,  189,  212,  213,  218,  221,  231, 
239,  256,  270,  274,  275,  281,  291, 
298,  306,  308,  309,  361,  385,  466, 
479,  493,  503,  513,  616.  See  also 
Gall-stones.     See  also  Lipoids. 

calculi,  463 

esters,  69 

in  effusions,  357 

pathological  occurrence  of,  419-421 
Cholesterolemia.  420,  491,  494,  555 
Chohne,  23,  73,  118,  360,  507,  570,  593, 

616 
Chondrodystrophia   foetalLs,    609 
Chondroid,  421 
Chondroitin-sulphuric    acid,    422,   431, 

465,  517 
Chondroma,  517 
Chondrosin,  422,  520 
Chromatin,  21,  40,  94,   101,   103,  108, 

369,  377 


Chrome  reaction,  015" 
Chromium,  4S2 
Chrom()[)hile  ccIIh,  480] 
Chyliform  fluids,  303 
Chylothorax,  304 
Chylous  cfFuBions,  363-366 
Chyluria,  364 
Ciaccio's  method,  408 
Circulation,  disturbance  of,  290  329 
Cirrhosis,   71,   72,    80,    321,    3.50,    570, 
594 

atrophic,  506 
Cloudy  swelling,  396-399 
Co-agglutination,  183 
Coagulation,  30 

necrosis,  382 

of  blood,  140,  201,  299,  493 

reaction  in  syphilis,  233 

time,  322,  295 
Coagulin,  316,  317,  324,  383 
Coal-pigment,  469 
Cobra  venom,  143,  223,  224 
Coelenterates,  158 
Coenurus.  132 
Coenzymes,  55 
Cold,  effects  of,  373 
Colloid,  29,  31-39,  160,  175,  181,  185, 
208,  218,  459 

carcinoma,  523 

degeneration,  429-430 

hydration  capacity  of,  346 

osmotic  pressure,  34 

precipitation  of,  30 

structure  of,  37-39 

thyroid,  430,  600-601 

water  absorbing  capacity,  336 
Colloidal  chemistry,  450 

solutions  of  metals,  50 
Colon  bacillus,  89,  104,  111,  126,  583, 
585,  589 

pigmentation  of,  478 
Colostrum,  215 
Colubridte,  142 
Coma,  diabetic,  558-563 
Complement,  207-209,  214,  229 

deviation,  229 

fixation  reaction,  131,  229-233 

hemolytic,  216-218 
Complementoid,  209 
Concretions,  452-470 

cutaneous,  408 

intestinal,  466 

preputial,  407 

prostatic,  407 

tonsillar,  408 

urinary,  459-464 
Congenital  edema,  352 
Congestion,  passive,  71 
Conglutination,  183 
Conglutinin,  218 
Copepods.  249 
Copper,  240 

Corpora  amylacea,  464,  407 
Corpus  luteum,  23,  276,  615 


684 


INDEX 


Corpuscles,  agglutination  of,   218-219 

fragility  of,  227 

stroma  of,  210-216 
Cotyledon  Scheideckeri,  220 
Crabs,  157 
Creatine,  79,  86,   132,    295,    395,    568, 

623 
Creatinine,  79,  501 
Cresols,  242,  583 
Cretinism,  607-609 
Crotalus,  142 
Crotin,  138 
Croton  tiglium,  138 
Crystalloids,  24-31 
Culex  mosquitos,  154 
Curcin,  138 

Cutaneous  concretions,  468 
Cyanosis,  enterogenous,  486,  589 
Cyclamin,  222 
Cyclic  vomiting,  568 
Cysteine,  244 
Cysticercus,  132 
Cystine,  288,  590-592 

calculi,  463 
Cystinuria,  590-592 
Cysts,  butter,  522 

dermoid,  521 

oil,  522 

ovarian,  519-522 

soap,  522 
Cytolvsis,  209 

of  tissue  cells,  233-236 
Cytoplasm,  42-45,  379 
Cytosine,  627 
Cytotoxins,  209-210 
Cytozyme,  316 

Daboia  Russellii,  148 
Danysz  effect,  175 
Dark  field  illumination,  212 
Deaminizing  enzymes,  78 
Death.     See  Necrosis. 
Decomposition  of  fats,  592 
Defensive  ferments,  204 
Deficiency  diseases,  280-289 

rickets  as,  288 
Degeneration,  colloid,  429-430 

fatty,  230,  275,  400-421 

hyaline,  427-429 

liver,  95 

mucoid,  430-432 

nerve,  87 

parenchymatous,  396-399 

reaction  of,  395 

waxy,  197,  394 
of  muscles,  395 
Dehydration,  245 
Delirium  tremens,  407 
Dementia  prjBCOx,  227 
Dercum's  disease,  518 
Dermoid  cysts,  521 
Desoleolecithin,  223 
Diabetes,  65,  71,  74,  360,  415,  421,  623, 
642-678.     See  also  Glycogen. 


Diabetes,     bronzed,     488.      See     also 
Hemochroma  tosis. 

glycogenic  infiltration  in,  437 

insipidus,  623 

kidney,  649 

lipemia  in,  72 

pancreatic,  671-678 

phlorhizin,  498,  666-671 
Diabetic  coma,  558-563 
Diacetic  acid,  558-563 
Diamines,  589 
Diapedesis,  294 
Diastase,  49,  55,  57,  61,  73-74,  272,  292, 

362,  390 
Dibothriocephalus  latus,  132 
Diet  and  tumor  growth,  513 
Diffusion,  26-31,  211 
Digitonin,  221 
Dihydroxyacetone,  653 
Di-iodotyrosine,  601 
Dilatation  of  stomach,  595 
Dimethylamine,  117 
Diose,  652 
Diphtheria,  383 

bacillus,  104,  105,  106,  125 

toxin,  69,  71,  81,  97,  173,  177,  253 
Dissociated  jaundice,  494 
Dittrich's  plugs,  391 
Dopa-oxidase,  67,  473-476 
Dracunculus,  137 
Dropsy,  soda,  351 
Ductless  glands,  597-625 

Earthworms,  137 

Echinococcus,  131,  162 

Echinoderms,  164 

Eclampsia,   65,   72,   96,    322,   541-547, 

577 
Edema,  330-336 

angioneurotic,  352 

cardiac,  348 

congenital,  352 

ex  vacuo,  342,  384 

fluids,  composition  of,  352-365 
physical  chemistry  of,  354 

hereditary,  352 

in  inflammation,  351 

neuropathic,  351 

nutritional,  285 

osmotic  pressure  in,  345 

renal,  349 
Edestin,  161 
Eel  serum,  158,  224 
Effusions,  cholesterol  in,  357 

meningeal,  359-362 

proteins  of,  356 

subcutaneous,  359 
Egg  proteins,  166 

sea-urchin,  64 
Ehrlich's  theory,  123-124,  172-174 
Eikosyl  alcohol,  521 
Elastin,  423 
Elastometer,  348 
Electric  shock,  379 


INDEX 


685 


Electrical      cuiuhictivity,      270,     371, 

534 
Electricity,  379 
Electrolytes,  26-31,  32 

of  exudates,  355 
Eledone  moschata,  164 
Elephantiasis,  340 
Ellagic  acid,  467 
Embolism,  325-327 

air,  326 

fat,  325,  416 
Emphysematous  gauRrene,  392 
Emulsin,  50,  55,  57,  61 
Emulsions,  32 
Endolysins,  207,  259 
Endotheliolvtic  serum,  235 
Endotheliotoxin,  147,  235,  294 
Endothclivun,  93 

Endotoxins,  98,  114,  124-125,  201 
End-piece,  209,  217 
Enols,  659 

Enterogenous  cyanosis,  486,  589 
Enterokinase,  55,  389 
Enteroliths,  466 
Enzymes,  48-100,  121,  208,  292,  358 

action,  principles  of,  50-54 

activation  of,  55 

antisera,  57-62 

bacterial,  109-115 

immunity  against,  112 

deaminizing,  78 

glycolytic,  70 

in  venoms,  144 

intracellular,  62 

nature  of,  49-50 

of  purine  metabolism,  630-633 

of  tumors,  505 

oxidizing,  62,  63-70 
of  bacteria,  112 

peptolytic,  79,  85,  88 

properties  of,  54 

proteolytic,  75-100,  111 
of  leucocytes,  89-90 

purine,  502 

reducing,  67 

resemblance  to  toxins,  62 

reversible  action,  51-54 

toxicitv  of,  55-57 
Eosin,  143,  376 
Eosinophiles,  69,  312,  434,  480 
Eosinophilia,  128,  198,  363,  421,  436 
Epeira  diadema,  152 
Epilepsv,  120,  360,  570 
Epinephrine,    155,   164,  475,  476,  480, 
584,  609,  615-621,  666 

arterial  degeneration  from,  619 
Epiphanin  reaction,  190 
EpitheHolysin,  236 
Erepsin,  71,  75,  84,  90,  110 
Ereptase,  78,  83,  99,  506,  554,  573 
Ergot,  585 
Erysipeloid,  157 
Erythrocytes,  resistance  of,  510 
Erythrocytolysis,  210-228 


Estern.sc,  50,  70,  71 
Ethereal  sulpliuric  acid,  580 
Ethyl  alcohui,  SO 

m('rca|)t;in,  590 

Hulidudf,  .")'.»() 
Fit hvlidi'iidiii mine,  590 
Euglohulin,  176,  1S6,  209,  351 
Exophthalmic  goiter,  73,  236,  609-612, 

614 
Exudates,  72 

autolysis  in,  88-89 

dilTcrciitiation  from  transudates,  363- 
358 

electrolytes  of,  355 

freezing  point  of,  355 

glycogen  in,  437 

immune  bodies  in,  358 

tuberculous,  363-368 

Famine  edema,  285 
Fat,  2223 

bacterial,  105-107 

content  of  liver,  406 

decomposition  of,  592 

embolism,  325,  416 

masked,  22 

metabolism,  52 

necrosis,  387-391 

of  bacillus  tuberculosis,  105-107 

physiological  formation  of,  401 

serous  atrophy  of,  342,  395 

soluble  vitamines,  462 

stains,  403 

sugar  from,  670 
Fatigue,  119,  569-571 

poison,  135 

toxins,  69 
Fatty  acids,  69,  134,  208,  226,  230,  242, 
270,  272,  274,  325,  387,  403,  589, 
592 
pathological  occurrence  of,  418 

degeneration,  230.  275,  400-421 

infiltration,  400-421 

metamorphosis,  400  421 
lipoids  in,  406-408 
Fecal  stones,  467 

thrombosis,  325 
Fermentation,  alcoholic,  53 

of  carbohydrates,  592 
Ferments,  defensive,  204 
Fcrratin,  484 
Fetal  bronchiectasis,  363 
Fetus.  139,  202,  215.  371,  393.  420,  433, 
545.  568,  693,  617 

autolvsis  of,  93,  97 
Fever,  21,  77,  87,  397,  568 

blackwater,  226 
Fibrin  calculi,  463 

ferment,  61,  272,  296.  299,  316 

formation,  316-325 
Fibrinogen,    291,    295.    296,    309,    315, 

321,  351,  361.  383,  554 
Fibrinolvsin,  112 
Fibrinolysis,  86,  92,  316,  321,  323,  325 


686 


INDEX 


Fibroma,  516 

Filaria,  137 

Fish,  poisonous,  156-158 

Flagella,  107 

Flies,  black,  154 

Fluorides,  240,  381 

Food  hormones,  280-289 

poisoning,  117 
Formic  acid,  153,  351 
Fragility  of  corpuscles,  227 
Freezing,  212 

point  of  exudates,  355 
Freund-Kaminer  reaction,  515 
FroeUch's  syndrome,  622 
Frog  corpuscles,  224 

poisons  of  dermal  glands,  155 
Froin's  syndrome,  362 
Fructose,  662 
Fuchsin  bodies,  429,  434 
Fusible  calculi,  462 

Galactose,  661 
Gall-bladder,  hydrops  of,  363 
Gall-stones,  453-458 
Gangrene,  91,  117,  391-392 

carbolic  acid,  380 

emphysematous,  392 

gas,  252,  568 

pulmonary,  273 
Gar,  157 
Gas  gangrene,  252,  568 

illuminating,  69 
Gastric  dilatation,  595 
Gastro-intestinal  autointoxication,  574- 

586 
Gaucher's  disease,  407.  421 
Gelatin,  20,  34,  37,  192,  320,  322,  336, 

423,  581 
Gelatinase,  111 
Gels,  32 

Gentisic  acid,  588 
Geotropism,  249 
Giant-cells,  69 

formation  of,  257,  265 
Giantism,  609 
Gila  monster,  149 
Glabrificin,  180 
Gland,  mammary,  82 
Gliadin,  277,  280,  302 
Globin,  20,  161,  192,  225,  296,  481 
Globulins,  21,  184,  193,  209,  216,  231, 

232,  355,  372 
Gluco-protein,  21 
Glucosamin,  105,  274,  430 
Glucose  metabolism,  642-678 
Glucoside,  141,  143,  161,  162,  221,  222 
Glucothionic  acid,  271 
Glutamine,  580 
Glutin-casoin,  251 
Glycerol,  251 
Glycerose,  653 

Glycine,  20,  112,  251,  280,  535,  580,  638, 
641,  653 

as  protective  substance,  243 


Glycogen,  23,  73,  74,  78,  105,  128,  132, 
135,  228,  240,  251,  417,  432-438. 
See  also  Diabetes. 

in  animal  parasites,  436 

in  exudates,  437 

in  leucocytes,  436 

in  tumors,  435,  501 
Glycogenic  infiltration,  434-438 

in  diabetes,  437 
GlycoUic  aldehyde,  652 
Glycolysis,  70 
Glycolytic  enzymes,  70 
Glyco-nucleoproteins,  104 
Glycoprotein,  21 
Glycosuria,  623,  664-666 
Glycothionic  acid,  427 
Glycuronic   acid,    239,   283,    512,    580, 
583,  666 
as  protective  substance,  243 
Glycylglycine,  88,  192 
Glycyl-tryptophane,  78 
Goiter,  chemistry  of,  604-607 

exophthalmic,  73.  236,  609-612,  614 
Goldzahl,  361 
Gorgonin,  601 
Gout,  310,  626-641 

calcium,  443 
Gouty  deposits,  468 
Grani  staining,  102,  106,  108 
Grammacese,  203 
Granulation  tissue,  278 
Granules,  Altmaim's,  93 
Granulomas,  castration,  278,  519 
Growth,  624 

chemistry  of,  276-280 
Guanidine,  135,  614 

methyl,  614  " 

Guanine,  627 
Guanylic  acid,  630 
Gum,  bacterial,  105 

Hairless  pig  malady,  607 
Haptophore,  123,  172 
Hay-fever,  203 
Heat.     See  also  Burns. 

effect  on  tissues,  372 

rigor,  372 
Hedera  helix,  222 
Heliotropism,  249 
Helminths,  133 
Heloderma  suspectum,  140 
Helvella  esculenta,  140,  222 
Helvellic  acid,  222 
Hemagglutination,  325 
Hemagglutinin,  133,  147,  218-219 
Hematin,  291, 296,  306, 478, 481-488, 489 
Hematinemia,  482 
Hematoidin,  296,  391,  476,  478,^,481- 

488,  489 
Hematoporphyrin,  484 
Hemicellulose,  105 
Hemiplegia,  421 
Hemochromatosis,  485,  487 
Hemochroniogen,  291,  206,  481 


INDEX 


087 


Hemocjanin,  KiO 
Hemofuscin,  486 

HemoKlohin,    20,    211,    21(),    22.'),    277, 
291,  296-298,  302,  481-488 

infarcts,  222 
HemoKlobinomia,  22.5 
HemoKlobimiria,    130,    220,    222,    225, 
303,  481 

paroxysmal,  227-228 
Hemolvniph  filaiids,  226 
Hemolysin,  147,  151,  152,  156 

sheep  corpuscle,  167 
Hemolysis,    72,    100,    110,    132,    154. 
210-228,  220,  303,  306,  307,  308, 
482,  490,  493.  496,  571 

by  bacteria,  219-220 

by  saponins,  221-223 

by  vegetable  poisons,  220-223 

by  venoms,  223-224 

in  cancer,  .509 

in  disease,  224-228 

pathological  anatomy,  228 

physical  chemistry  of,  212 

postmortem,  219 

resistance  to,  227 
Hemolytic  amboceptor,  215-216 

complement,  216-218 

icterus,  494 

jaundice.  225 
Hemophilia,  298-300 
Hemorrhage,  169,  293-300 

cerebral,  297 
Hemorrhagic  infarcts,  328 
Hemorrhagin,  147,  235 
Hemosiderin,  296,  329,  483-488 
Hemotoxins,  147,  220 
Heparin,  316 
Hereditary  edema,  352 
Heroine,  238 
Herpes  zoster,  268 
Heterolysis.  85 
Hexoses,  657-661 
H-ion  concentration,  212 
Hippuric  acid,  243,  368 
Hirschfold  and  Klinger  reaction,  233 
Hirudin.  320 
Histamine,  154,  196,  584-586,  596,  616, 

622 
Histidine,  91,  584 
Histon,  45,  160,  192,  310,  390,  423 
Hodgkin's  disease,  65,  312 
Homogentisic  acid,  67,  477,  586-589 
Hornets,  154 
Hyaline  degeneration,  427-429 

thrombi,  324,  374,  380 
Hydration  capacity  of  colloids,  346 
Hydroa  aestiva,  485 
Hydrocele,  359 
Hydrocephalus,  360 
Hydrochinon.  583 

reaction,  68 
Hydrogen,  arseniuretted,  213 

sulphide,  590 
Hydrolysis,  19 


Hydrophinai,  142,  144 
Hydrops  of  gall-bludder,  MV.\ 
Hydroquinone,  17H 
Hydroxylamine,  5.S9 
Hydroxy  stearic  acid,  413 
Hypercholesterolemia,  457 
Hyperemia,  312  316 
Hypernephroma,  '.ft,  524 
Hyperthyreosis,  65 
Hypertrophy,  27.S 
Hypophysis,  277,  621-624 
Hypoxanthine,  627 

ICHTHYOTOXIN,   158,  224 
Icteric  necrosis,  493 
Icterus,  222,  225,  227,  293,  303,  .321, 
322.  488-496 

dissociated,  494 

hemolytic,  225,  494 

neonatorum,  491 
Idiosyncrasy,  237 

Ileus,  594.     See  also  Intestinal  obstruc- 
tion. 
Illuminating  gas,  69 
Imbibition,  34 
Immune  body,  206 
in  exudates,  358 

reactions,  specificity  of>  165-172 
Immunity,  159-246 
Immunity  against  bacterial  enzymes,  112 

in  unicellular  organisms,  246 

reactions  in  cancer,  514-516 

to  phytotoxins,  139 
Inanition,  568 
Indican,   362,  463,  510,  580,   581-583, 

596,  607 
Indicanemia,  537 
Indigo  calculi,  463 
Indole,   242,   274.   473,   498,   500,   569, 

579,  580,  581-583,  594 
Indole-acetic  acid,  579,  583 
Indophenol  reaction,  69 
Indoxyl,  242,  580 
Infancy,  acidosis  in,  566 
Infantilism,  intestinal,  595 
Infarction,  327-329 
Infarcts,  86 

hemoglobin,  222 

hemorrhagic,  328 

uric-acid,  640 
Infection,  autolysis  in  relation  to,  97 
Infectious  diseases,  SO 
Infiltration,  fatty,  400-421 

glycogenic,  434-438 
in  diabetes,  437  | 
Inflammation,  247-272 

edema  in,  351 
Infusoria,  129.  250 

Inorganic     poisons,     defense     against, 
240-247 

substance  of  cells,  24 
Inosite,  132 

InsectS;  poisons  of,  163 
Insolation,  374 


688 


INDEX 


Intercellular  substance,  47 
Intermediary  body,  207 
Intestinal  concretions,  466 

infantilism,  595 

obstruction,  581,  596 

sand,  467 
Intoxication,  acid,  555-569 
Intracellular  enzymes,  62-100 
Invertase,  49,  55 
Invertin,  61 
Iodides,  65 

lodin,  163,  170.     See  also  Thyroid. 
Iodized  proteins,  601 
Iodoform,  94,  163,  268 
lodophilia,  437 
lodothyrein,  600 
Ionization,  25  ' 

Iron,  297,  302,  303,  306,  308,  329,  472, 
483,  487,  504 

in  calcification,  440 

pigments,  484 
Iron-lime  deposits,  441 
Iron-proteins,  24 
Isaria  densa,  112 
Isoagglutination,  219,  226 
Isoamylamine,  118,  589 
Isobutylamine,  584 
Isocetinic  acid,  106 
Isohemolysins,  215 
Isohemolysis,  226 
Isonephrotoxins,  235 
Isoprecipitins,  184 
Ivy,  poison,  222 

Jatropha  curcus,  138 
Jaundice,  222,  225,  227,  293,  303,  322, 
488-496 

dissociated,  494 

hemolytic,  225,  494 

in  newborn,  499 
Jecorin,  291 
Jequirity,  251 

KAMiNER-Freund  reaction,  515 
Karyokinesis,  40,  279 
Karyolysis,  94,  368 
Karyorrhexis,  94,  368 
Kenotoxin,  570 
Keratin,  194 
Keratohyalin,  429 
Keratomalacia,  284 
Kidney  diabetes,  649 
Kinases,  55 

Klausner's  serum  reaction,  233 
Krait,  148 
Kynurenic  acid,  580 

Laccase,  61,  67 

Lactalbimiin,  184 

Lactarius  torminosus,  141 

Lactic  acid,  79,  89,  91,  96,  100,  208,  251, 
272,  394,  401,  501,  544,  552,  564, 
592,  653.     See  also  Diabetes. 
from  triose,  655 

Lactosuria,  663 


/ 


Laking  of  blood,  211 
Lamprey  serum,  158 
Landau's  reaction,  233 
Lange  reaction,  37 
Lanthanin,  40 
L-arabinose,  105 
Lathy  rism,  138 
Latrodectes  mactans,  152 

tredecimguttatas,  152 
Laurie  acid,  106  / 

Lecithids,  223 
Lecithin,  22-23,  79,  95,  118,  131,  163, 

208,  212,  215,  218,  221,  223,  230,  256, 

257,  270,  274,  275,  291,  306,  309,  357, 

369,  504,  553,  593.     See  also  Lipoids. 
Lecithinase,  71,  507 
Lens  proteins,  166 
Leucine,  88,  91,  95,  111,  251,  271,  310, 

357,  391,  544,  550-555,  589,  591 
Leucineimid,  641 
Leucocidins,  234 

Leucocytes,  64,  68,  69,  73,  78,  82,  85, 
207,  291,  214,  215,  309.  See  also 
Inflammation. 

bactericidal  substances  of,  259 

glycogen  in,  436 

proteolytic  enzymes  of,  89-90 
Leucocytolysins,  147 
Leucocytolysis,  214 
Leucocytolytic  serum,  234 
Leucocytotoxin,  234,  311 
Leukemia,   68,   89,   293,   309-312,   321, 
378,  436,  577 

autolysis  in,  98 
Leukoprotease,  90,  270 
Levulose,  662 
Levulosuria,  662 
L-fructose,  653 
L-glyceric  aldehyde,  653 
Lice,  154 

Light,  effects  of,  374 
Linin,  40,  104 
Lipase,  51,  52,  56,  57,  61,  70-73,  90,  98, 

110,  113,  132,  163,  217,  218,  223,  270, 

272,  326,  331,  358,  387,  411,  417,  507, 

518,  543,  554,  603 
Lipemia,  295,  415-418 

in  diabetes,  72 
Lipins,  22-23,  107,  162,  357 

of  tumors,  503 
Lipocyaiiin,  127,  479 
Lipochrome,  106,  127,  281,  478-480 
Lipofuscins,  478 
Lipoidaso,  73,  90 
Lipoidcmia,  416 

Lipoids,  22-23,  43,  45,  59,  70,  81,  113, 
122,  163,  185,  213,  217,  226,  230, 
306,  308,  318,  465,  599.  See  also 
Cholesterol  and  Lecithin.  See  also 
Lipeniia. 

as  antigens,  162-163 

doubly  refractive,  23 

in  fatty  metamorpliosis,  406-408 

of  adrenal,  ()16 


jM)i':x 


689 


Lipoma,  518 
Lip<)-s:irc(tiiia,   aH' 
Liposomes,  404 
Liquefaction  necrosis,  383 
Lithofellic  acid,  407 

Liver,  acute  vollow  atrophy  of,  ".).),  047- 
555,  hli 
autolysis  of,  93,  95 
dep;eneratioiis,  95 
fat  content,  400 
Lobster,  L57 
Iv-sorbose,  05:^ 
Lumbricin,  137 
\Ain\i  stones,  468 
Lutein,  479 

Lymph,  absorption  of,  338 
composition  of.  330 
formation  of,  331-338 
Lvmphagogues,  332 
Lymphatolytic  serum,  23o 
Lvmph-dands,  72,  97 
Lvmphocvtes,  72,  90,  98,  217,  232,  2o3, 

'254 
Lymphocytosis,  376,  378 
Lymphoid  cells,  71 

tissues,  90 
Lymphosarcoma,  499 
Lysine,  237,  583 
Lysol,  239,  583 

Magnesium,  256 
Malaria,  130,  225,  226,  482 
Malarial  pigmentation,  478 
Malic  acid,  248 
Malmignatte,  152 
Maltase,  51,  98 
Maltosuria,  664 
Mammary  gland,  82 
Manganese,  81 
Margarin,  391,  418 
Masked  fat,  22 
Mast  cell,  42,  45,  254,  310 
Mechanical  injury  of  cells,  381 
Megakarocytes,  69 
Mehlnahrschaden,  286 
Meiostagmin  reaction,  189 
Melanin?  67,  164,  188,  471-478,  487 
composition  of,  472 

Melanosarcoma,  67,  474 

Melanotic  tumors,  474 

Melanuria,  474 

Melena  neonatorum,  29S,  .5^21  ^ 

Membranes,  semipermeable,  2  / 

Meningeal  effusions,  359-362 

Meningitis,  360 
tuberculous,  361 

Menstrual  blood,  321 

Mercury,  25,  80,  240,  550 

Mesoporphyrin,  484 

Metabolism  53 

abnormalities  m,  530-69b 
after  parathyroidectomy,  61^ 
basal,  602,  607,  623,  638 

44 


Metabolism,  ealrium.    S«>i-  (httomalacia 
:in<l  /('(<•A•'^^•• 
riirltolivdnite,  642  -678 
fat,  52' 

glucose,  642  678 
in  unaphyi.'ixis,  198 
in  cancer,  510 
in  sup|>ur,itic)n.  272 
influence  of  thvroid.  697 
purine,  626  641 

enzvmes  of,  630  633 
relation  of  autolysis  to,  81   82 
sulnhur.  in  cancer,  511 
Metalbumin,  521 
Metals,  colloidal  solutions  of,  ;><• 
Metamorphosis,    fattv,    400  421 

lipoids  in,  406  408 
Mctapla.sia.  279  , 

Metastatic  ealcification,  442,  oJo 
Methane,  244 

Methemoglobin,  220,  29S,  486 
Methvlamine,  117 
Methvlation,  241,  244 
Methylene  blue,  67 

reduction  of,  68 
Methvl  guanidine,  196.  5/3  t.M 
Methvl  mercaptan,  582,  59U 
Mid-piece,  209,  217 
Mitochondria,  44,  397,  408,  o03,  <.0., 
Molds,  107 
Molluscs,  164 
Monobromacetic  acid,  6\fo 
Moray,  158 

Morbus  maculosus,  484 
Morchella  esculenta,  223 
Morner's  body,  310,  526 
Morphine,  69 
tolerance,  238 

^S:^'Kmy3N57,430  432, 

517,  519-520,  60S 
Mucoid  degeneration,  430 -4i/ 
Mucoids,  519  520 
Mucoitin-suliihuric  acid,  l.U 
Multiple  myeloma,  525 

sclerosis,  418 
Mummies,  54,  184,  194,  414 
Mura'na  helena,  158 
Mursnida^,  156 
Muscarine,  lis   119,  141.  n9.i 
Muscles,  waxy  degeneration  of,  390 
Mushrooms,  547 
poisonous,  222 
poisons,  140-141 
Myelins,  23,  93,  397,  39s,  407 
Myeloblasts,  90 
Myelocj-tes,  69,  89 
Myelomas,  90 

multiple,  525 
Myelopathic  albumosuria,  526-529 
Myelotoxic  serum,  235 
MVkol,  106 
Myocardium,  22,  2/8 
Myoma,  516 


690 


INDEX 


Myosin,  393 
Myristic  acid,  521 
Myristinic  acid,  106 
Myrosin,  55 
Myxedema,  607-609 
Myxoma,  517 

Naphthalin,  243 
Naphthvlaminol,  498 
Nastin,162,  163 
Necrosis,  367-392 

autolysis  in,  92-94 

chloroform,  321 

coagulation,  382 

fat,  387-391 

liquefaction,  383 

pancreatic  fat,  387-391 
Necrotic  areas,  autolysis  in,  92-94 
Necturus,  149,  224 
Neisser-Wechsberg  phenomenon,  229 
Nematodes,  134-137 
Nephritis,  65,  72,  74,  93,  226,  293,  349, 
358,  416,  420,  577,  617,  640.     See 
also  Edema.     See  also  Uremia. 

acidosis  in,  565 
Nephrolytic  serum,  235 
Nerve  degeneration,  87 

section,  395,  396 

tissues,  118 
autolysis  of,  93 
Neurine,  118,  119,  570,  593 
Neuritis,  experimental,  281 
Neurolytic  serum,  235 
Neuropathic  edema,  351 
Neurotoxins,  147,  235 
Nicotine,  619 
Nicotinic  acid,  282 
Ninhydrin,  204 
Nissl  bodies,  45,  146 
Nitrites,  486 
Nitrobenzol,  213,  486 
Nitrophenols,  547 
Nitroprotein,  170 

Non-protein  antigens,  161-165  < 

Nuclease,  79,  92,  99,  110,  111,  369,  506, 

630 
Nuclei,  structure  and  chemistry  of,  40- 

42 
Nucleic  acid,  21,  40,  92,  111,  121,  162, 

192,  236,  369,  502,  629-631 
Nuclein,  21,  94,  104,  114,  189,  240,  270, 

274,  278,  499 
Nucleohiston,  274,  499 
Nucleoli,  41 
Nucleo-protcins,  21,  24,  40,  42,  50,  64, 

79,  98,  103,  108,  125,  167,  192,  216, 

234,  239,  271,  278,  291,  310,  324,  357, 

369,  446,  499,  600,  629-631 
Nucleotids,  629 
Nucleus,  379,  381 
Nutritional  dropsy,  286 

Obstruction,  intestinal,  581,  596 
Ochronosis,  476-478 
Oestrus  equi,  307 


Oil  cyst,  522 

Oleic  acid,  133,  223,  414,  545 
Ophidia,  141 
Ophiotoxin,  143 
Opsonins,  163,  188-189,  258 
Oral  mucosa,  pigmentation  of,  478 
■Organ  extracts,  toxicity  of,  197 
Organic  poisons,  defense  against,  241 
Ornithine,  118,  590 
Ornithorhvnchus  paradoxus,  150 
Osmic  acid,  216,  403 
Osmosis,  26-31,  211 
Osmotic   pressure,    29,    334,    337,    371, 
382,  398,  534.     See  also  Edema. 
in  edema,  345 
of  colloids,  34 
Ossification,  439,  624 
Osteitis  deformans,  449 
Osteogenesis  imperfecta,  451 
Osteohemachromatosis,  478 
Osteomalacia,  447-449 
Ovarian  cyst,  519-522 
Ovomucoid,  160,  192 
Oxalic  acid,  69,  381,  462,  592,  633 
Oxidases,   50,   59,   63-70,   90,   98,   242, 
272,  358,  411,  472 

alcohol,  66 

reaction,  310 

xanthine,  66 
Oxidation,  41,  241,  247,  552 

as  defense  mechanism,  242 

by  enzymes,  63-70 
Oxidizing  enzymes,  62,  63-70 

of  bacteria,  112 
Oxygenase,  66 
Oxymandelic  acid,  551 
Oxy-proteic  acid,  511 

Pancreas,  self-digestion  of,  391 
Pancreatic  calculi,  465 

diabetes,  671-678 

fat  necrosis,  387-391 
Pancreatin,  56 
Pancreatitis,  387-391,  73 
Papain,  56,  97 
Papayotin,  251 
Parachromatin,  40 
Paracresol,  579,  583 
Parahemoglobin,   298,   481 
Para-hydroxy-phenyl-cthylaminc,  584 
Paralbumin,  521 
Paralinin,  40 
Paralysis  agitans,  614 
Paramecia,  246 
Paramecium,  601 
Paramucin,  431,  520 
Paranuclcin,  161 

Para-oxyplicnyl  acetic  acid,  579,  583 
Para-oxy  phenyl-propionic     acid,     579, 

583 
Paraphenylamine,  584 
Parasites,  animal,  128-137 
chemistry  of,  128-137 
glycogen  in,  436 


INDEX 


O'J  1 


Parathyroid,  613  615 

antis(>rum,  2;ir) 
Parathvroiclectomy,   nictaholisin   after, 

013  " 
Parenchymatous  doRC'iK-ration,  396  399 
Paroxysmal  hemoglobinuria,  227  228 
Parrot  fish,  157 
Passive  anaphylaxis,  201 

congestion,  71 
Pellagra,  287 
Pentosan,  105 

Pentose,  99,  104,  390,  502,  553,  656-657 
Pentosuria,  657 

Pepsin,  49,  55,  56,  57,  58,  61,  97,  575 
Peptase,  83 
Peptids,  78,  99,  192 
Peptolytic  enzvmes,  79,  85,  88 
Peptones,  87,  88,  91,  98,  102,  115,  135, 

185,  251,  270,  271,  274,  310,  319,  332, 

391,  427,  585,  575-578 
Permanganate  reduction  index,  361 
Permeability  of  capillaries,  333,  342 
Pernicious  anemia,  227,  305-308,  478 

vomiting  of  pregnancy,  546 
Peroxidase,  66,  68,  69,  90,  507,  554,  603 
Peroxides,  64 
Persensitization,  218 
Pfeiffer  phenomenon,  207,  248 
Phagocytes,  74,  215 
Phagocytosis,  188,  247,  248,  267,  314 
Phallin,  141,  221,  222 
Phallusia  mamillata,  164 
Phanerosis,  404 
Phaseolus  multiflorus,  218 
Phenol,  242,  380,  477,  510,  569,  579,  583 
Phenolase,  67,  79 
Phenvlacetic  acid,  580,  583 
Phenylalanine,  472,  579,  584,  586-589 
Phenyl-ethylamine,  584 
Philocatalase,  64 
Phlogosin,  250 
Phlorhizin,  96 

diabetes,  498,  666  671 
Phosphate  calculi,  462 
Phosphates,  565 
Phosphatids,   22,    106,    162,   281,    316, 

479 
Phospho-glycoprotein,  21 
Phospholipins,  22,  29,  44,  45,  279,  295, 

316,  385 
Phosphoprotein,  21 
Phosphoric  acid  in-  calcification,  446 
Phosphorus,  81,  95,  241,  405,  451,  547 

poisoning,  65,   68,  71,  95,  435,  548, 

552,  577,  661.     See  also  Fatty  meta- 
morphosis. 
Physical  chemistry  of  cell,  24-39 
of  edema  fluids,  354 
of  hemolysis,  212 
Physics,  cellular,  17-47 
Phyto-precipitins,  187 
Phytotoxins,  138-141 

immunitj^  to,  139 
Picric  acid,  550 


PiKmontatioii,  471  496 

nialariiil,  478 

of  colon,  478 

of  oral  mucosa,  478 
l'i>i;mcn(s,  b.-ictr-rial,  126-127 

bile,  488^  496 

blood,  481  488 

iron,  4.SI 

of  Addison's  disease,  475 

plant,  479 

waste,  478 
Pineal  gland,  624 
Piporazin,  312 
Pituitary  gland,  621-624 
Pituitrin,  622 
Placenta,  276.     See  also  Eclampsia. 

retention  of,  568 
Plant  cells,  28,  64,  373, 583 

pigments,  479 
Plasma  cells,  69 

fat  of,  291 
Plasmaphaeresis,  295 
Plasmase,  316 
Plasmodiimn  malaria?,  130 
Plasmolysis  of  bacteria,  101 
Plasmoptysis,  29 

of  bacteria,  101 
Plasmorrhexis,  29 
Plastein,  52,  100,  110 
Plastin,  40,  44 
Platelets,  316,  317,  323 
Platypus  venom,  150 
Pnein,  63 

Pneumobacillus,  251 
Pneumococcus,   70,   91,   114,   195,  220, 

270,  356,  486,  493 
Pneumonia,  57,  60,  65,  71,  273,  274,  293, 
320,  321,  322,  435,  568,  577,  616 

autolysis  in,  90-92 
Pncumonokoniosis,  469 
Pneumothorax,  chemistry  of,  365 
Poison,  bee,  153 

chemical  defense  against,  237  246 

fatigue,  135 

inorganic,  defense  against,  240-247 

mushroom,  140-141 

of  burns,  571-573 

of  dermal  glands  of  toads,  154 

of  insects,  153 

organic,  defense  against,  241 

protoplasmic,  380 

scorpion,  150 

spider,  152 

vegetable,  218 

hemolysis  by,  220  223 
Poisoning,  chloroform,  548,  567 

food,  117 

phosphorus,  65,  68,  71,  95,  435,  548, 
55:2,  571,  661 
Poisonous  bacterial  proteins,    126-126 

fish,  15t>-15S 

mushrooms,  222 
Pollen,  203 
Polycytiiomia,  313 


692 


INDEX 


Polyneuritis  gallinarum,  281 
Polypeptid,   19,   125,   160,   192,  511,  57 
Polyphenoloxidases,  66 
Porges-Hermann-Perutz  reaction,  233 
Porphyrins,  484 
Portuguese-man-o'-war,  158 
Postmortem  changes,  65 
autolysis  in,  96-97 

hemolysis,  219 
Potassium,  24,  306,  336,  360,  504,  532, 
535  542 

chlorate,  226,  325 

salts,  278 
Potato,  222 
Potocytosis,  333 
P-oxyphenyl-ethylamine,  308 
P-oxyphenyl-lactic  acid,  551 
Precipitation  of  colloids,  36 
Precipitinogen,  184 
Precipitins,  150,  184-188,  202,  358 
Precipitinoid,  186 
Precocity,  sexual,  615 
Pregnancy,  60,  71,  80,  204,  276,  457 

acetonuria  in,  568 

pernicious  vomiting  of,  546 

toxemias  of,  540-547 
Preputial  concretions,  467 
Pressor  bases,  584r-586 
Proliferation,  276-280 
Propionic  acid,  579 
Prostatic  concretions,  457 
Protamin,  20,  52,   126,   160,   161,  236, 

278,  390 
Proteases,  75-100,  113,  200,  292 
Proteic  acid,  307 
Protein,  94 

Bence-Jones,  525-529 

blood,  295 

chemistry  of,  19-22 

compound,  20 

egg,  166 

insoluble,  22 

iodized,  601 

lens,  166 

loss  in  sputum,  273 

of  effusions,  356 

of  sputum,  273 

of  tumors,  499-501 

poisonous  bacterial,  125-126 

pyogenic,  268 

racemized,  160,  165 

serum,  176,  315 

simple,  20 

sugar  from,  670 
Proteolysis,   165,   198,  201.  204,  217 
Proteolytic  enzymes,  75-l60,  HI,  129 

of  leu(;ocytes,  89-90 
Proteoses,  88,   115,   135,   143,   1(50,   192, 

201,  218,  274,  310,  319,  320,  327,  357, 

391,  427,  591,  551,  575-578,  596 
Prothr()ml)iii,  295,  298,  300,  303,  316 
Protoplasm,  18,  21,  35 

stru(;turo  of,  37-39 
Prot()])lasmic  poison,  380 


Protozoa,  chemistry  of,  129-131 
Pseudochylous  effusions,  364 
Pseudo-globulin,  176 
Pseudoleukemia,  312 
Pseudomelanosis,  485 
Pseudomucin,  430,  431 
Pseudo-myxoma  peritonei,  521 
Ptomains,"  116-120,  123,  391,  509 
Puerperal  eclampsia,  72,  96 

sepsis,  486 
Pulmonary  gangrene,  273 
Purine,  91,  369,  385,  499,  626-641 

bases,  79,  94,  310 

bodies,  272 

enzymes,  502 

metabolism,  enzymes  of,  630-633 

of  tumors,  502 
Purpura  hemorrhagica,  234,  298,  317 

neonatorum,  322 
Purpuric  diseases,  298 
Pus,  66,  68,  72,  74,  88,  94,  97,  267-273 

composition  of,  269-273 
Putrefaction,   111,   117,   177,   185,  209, 

391,  578,  581,  591 
Putrescine,  118,  589,  591 
Pycnosis,  92,  328,  369 
Pyin,  271,  426 
Pyocyanase,  115 
Pyocyaneus,  214 
Pyocyanolysin,  219 
Pyogenic  proteins,  268 
Pyopneumothorax,  365 
Pyridine,  241,  244,  282,  498,  528 
Pyrimidine,  282,  627 
Pyrocatechin,  583,  588,  019 

QUILLAJA,  221 

Quillajic  acid,  143 
Quinine,  226,  251,  256,  393 

Racemized  protein,  160,  165 
Radium,  81,  143,  99,  378,  311,  506 
Ragweed,  203 
Rana  esculenta,  155 
Rays,  serum  of,  158 
Reaction     of     blood,     292.     See     also 
Acidosis. 

of  degeneration,  395 
Receptors,  124,  172,  215 
Reducing  enzymes,  67 
Reduction  as  defense  meciianism,  241 
Regeneration,  276-280 

of  blood,  302 
Renal  edema,  349 
Rennin,  55,  57,  61,  110,  113,  272,  292, 

507 
Repair,  276-280 
Resistance  to  liemolysis,  227 
Resonance  theory,  170 
Retention  of  chlorides,  350 
Rhamnose,  056 
Hhinoliths,  468 
Rhumbler's  artificial  amcbao,  262 


INDEX 


693 


Rhus  divcrsiloha,  1 41 

toxicoclfMidron,  111,  Kil 
Ricin,  69,  72,  IC.l,  138  140,  221,  325 
Ricinus  conunuiiis,  1;}S 
Rickets,  449  452 

as  deficicncv  (lis(\ase,  288 
Rigor  mortis,  392 
Robin.  i;iS 

Robinia  pscudoucacia,  138 
Roentgen  rays,   1S5.     See  also  X-ray f<. 
Rovida's  hyalin  substance,  271 
Russell's  viper,  148 

Saccharosuria,  664 
Salamanders.  155 
Salicylic  aldehyde.  106 
Salivary  calcuh,  466 
Salmon.  278,  396 
Salts,  ammonium,  212 

bile,  214.  319.  322,  389,  453,  492-498 
Samandarin.  155 
Sand,  intestinal,  467 
Saponin,   143,  211,  212,  221-223,  227, 

239 
Saponins,  hemolysis  by,  221-223 
Sapotoxin,  222 
Sapremia,  392 
Sareocystin,  130 

Sarcolactic  acid,  542,  551,  564,  569 
Sarcoma,  69.  99,  504.  506,  525,  583 
Sarcosporidia,  128.  130 
Sclerema  neonatorum,  415 
Sclerosis,  multiple,  418  • 
Sclerostoma,  136 

equinum,  128 
Scolopendra  heros,  153 
Scorpion  poison,  150 
Scorpa^na  scorpha,  156 
Scurvy,  286,  298 
Sea-snake,  148 
Sea-urchin  eggs,  64 
Secondarv  anemia.  301-303 
Selenium^  81,  109,  241 
Semipermeable  membranes,  27 
Sepsis,  puerperal.  486 
Septicemia,  96,  219,  547 
Serosamucin,  357 
Serous  atrophy  of  fat,  342,  395 
Serozyme,  316 
Serum  albumin,  20 

anthracidal,  209 

antiplatelet,  234,  300 

antithyroid,  612 

eel,  158,  224 

endotheliolytic,  235 

foreign,  toxicity  of,  196 

Lamprey,  158 

leucocytolytic,  234 

lymphatolytic,  235 

myelotoxic,  235 

nephrolytic.  235 

neurolytic,  235 

proteins,  176,  315 

snake,  149 


Serum,  thvmotoxic,  230 

thyrolytir,  235 
Scxiial  prccocitv,  615 
Shock,  t;5,  ,S7,  200,  566 

electric,  379 
Sialolithiasis,  466 
Side  chain  theory,  124 
Siderosi.s,  469 
Silicates.  375,  441,  469 
Silicic  acid,  20,S,  21K 
Silver,  SO 
Sistrurus,  142 
Skatole,  242,  498,  500,  569,  580,  58.3 

acetic  acid,  579 
Skatoxyl,  242,  580 
Skepto-phylaxis,  197 
Snake  scrum.  149 

venoms,  141-150,  218 
Soap-l)ark.  221 

Soaps,  94,  97,  162,  213,  217,  223,  226, 
274,  275,  386,  387,  390,  403,  413. 
467 

calcium,  364,  445,  51S 

cysts,  522 
Soapstone,  469 
Soda  dropsy,  351 
Solanacea',218 
Sohmidin,  222 
Solanin,  222 
Specificity  of  immune  reactions,   166- 

172 
Spermatocele  fluid,  359 
Spermatotoxin,  236 
Spermatozoa,    40,    162,    167,  216,   24S. 

249,  278 
Spermin,  272,  312 
Spheroides  testudineus,  157 
Spiders,  221 

poison,  152 
Spina  bifithi,  360 
Spirochetes,  232 
Spleen,  83,  90,  95.  179,  185,  209,  226, 

369 
Splenectomy,  224 
Spongin,  691 
Spores,  107,  109 
Sputum,  88,  91,  273-275 

loss  of  protein  in,  273 

proteins  of,  273 
Squid,  67 

Staining,  Gram,  106,  108 
Stains,  fat,  493 

vital  45 
Staphylococcus,  86,  89,   112,   127.  219, 
479 

aureus,  107,  320 

pyogenes  aureus,  1 10 
Staphvlolvsin,  219 
Starch,  105,  110 
Starvation.  65,  82,  396,  567 

acidosis,  285 
Stone.     See  Calculus. 
Streptococcus,  86,  89,  110,  112,  486 

viridans,  220 


694 


INDEX 


Streptocolysin,  219 

Streptothrix,  162 

Stroma  of  corpuscles,  210-216 

Strontium,  614 

Struvit  stone,  462 

Subcutaneous  effusions,  359 

Succinic  acid,  132 

Succus  entericus,  56 

Sugar  from  fat,  670 

from  protein,  670 

in  blood,  649-652 
Sulphemoglobin,  486,  590 
Sulphhemoglobinemia,  589 
Sulphocyanide,  244 
Sulphonal,  485 
Sulphur,  244 

metabolism  in  cancer,  511 
Sulphuric  acid,  580 

as  defense  against  poisons,  242 
Sulphur-methemoglobin,  486 
Suppuration,  87,  267-273,  322,  358,  384, 
426,  577 

metabolism  in,  272 
Surface  phenomena,  50 

tension,  35,  181,  260-267 
Suspensions,  32 
Swelling,  cloudy,  396-399 
Synanceia  brachio,  156 
Syncytiolysin,  236,  544 
Synthesis  by  enzymes,  51-54 
SyphiUs,  72,  73,  224,  227,  228,  230,  361, 
550 

coagulation  reaction  in,  233 

Tactile  stimulation,  255 
Taenia,  131-134 

echinococcus,  131 

saginata,  131,  133 
Tarantula,  153 
Tartar,  466 
Taurin,  492 
Tellurium,  81,  109,  241 
Terpenes,  214 
Tetanolysin,  174,  177,  219 
Tetanus  toxin,  65,  69,  97,  122,  123,  173, 

598 
Tetany,  566,  595,  613-615 
Tethelin,  277,  513,  622 
Tetrodon,  157 
Tetrodo-toxin,  157 
Tetrose,  656 
Theobromine,  627 
Thermoprecipitins,  186 
Thermotaxis,  249,  254,  262 
Thermotropism;  249 
Thigmotaxis,  249 
Thorium,  207 
Tliorium-x,  61,  202,  378 
Thrombin,  316 
Thrombogen,  316 
ThrombokiiiJisc,  316 
Tliromboplastiii,  316 
Thrombosis,  315  325 

ferment,  ;}25 


Thrombus,  formation  of,   323 

hyalin,  324,  374,  380 
Thymine,  104,  627 
Thymotoxic  serum,  236 
Thymus,  94,  612,  624 
Thyreoglobulin,  600-601 
Thyroid,  65,  82,  597-612 
•   carcinoma,  508 

coUoid,  430,  600-601 

extract,  277 

influence  of,  in  metabolism,  597 
Thyroiodin,  600-601 
Thyrolytic  serum,  235 
Thyroxin,  601-602 
Tissue  cells,  cytolysis  of,  233-236 
Tissues,  effect  of  heat  on,  372 
Toads,  poisons  of  dermal  glands  of,  154 
Toluene,  80 
Toluylendiamine,  307 
Tonsillar  concretions,  468 
Tophi,  469 
Toxalbimains,  138 
Toxemias  of  pregnancy,  540-547 
Toxicity  of  foreign  serum,  196 

of  organ  extracts,  197 
Toxins,  97,  172-175,  376,  427 

bacterial,  120-125 

botulinus,  122 

diphtheria,  69,  71,  81,  97,  173,  177, 
253 

fatigue,  69 

resemblance  to  enzymes,  62 

tetanus,  65,  69,  97,  122,  123,  173,  598 
Toxoid,  212,  173 
Toxolecithid,  154 
Toxophore,  123,  172 
Trachinis  draco,  156 
Transudates,  88,  353 

differentiation   from   exudates,   353- 
358 
Trichinella  spiralis,  128,  135 
Triketohydrindene  hydrate,  204 
Trimethylamine,  117,  251,  593 
Trinitro-toluene,  550 
Triose,  lactic  acid  from,  655 
Trioses,  653 

Tropisms,  theory  of,  249 
Trypanosomes,  129,  246 
Trypsin,  49,  56,  57,  97,  187,  387,  575 

antiserum,  57-62 
Tryptophane,  280,  472,  474,  500,  576, 

579  581 
Tubercle  bacillus,  65,  72,  94,  102,  105, 
114,  125,  162,  163,320,  383,  384. 
See    also    Bacilliifi,     Tubercle. 
composition  of,  104-107 
Tuberculin,  81,  97,  120,  125,  167,  253, 

577 
Tubcrculonastin,  162 
Tubcrculosaniin,  384 
Tuberculosis,  60,  ()9,  71,  73,  89,  91,  94, 
96,  99,  2(iS,  271,  273,  271,  120,  434, 
435,  440,  485,  510,  577,  604.     See 
also  Caseation. 


IXDKX 


mri 


Tuhoroulosis,  bacillus  of.     Sec  Tubercle 
bdcillus. 

composition  of  tissues,  386 

exudates,  353-358 
Tuberculous  niouinnitis,  3G1 
Tumors,  (IS,  497  529 

autolysis  in,  98  100,  505 

benign,  ciicinistrv  ol",  516-522 

choniistrv  of,  497  -529 

cholestcatomatous,  41'.) 

enzymes  of,  505 

glycogen  in,  435,  501 

growth  and  diet,  513 

lipins  of,  603 

melanotic,  474 

proteins  of,  499-501 

purines  of,  502 
Turgor  of  plant  cells,  28 
Tvndall's  phenomenon,  33 
Typhoid,  65,  96,  309 

bacillus,  178,  183,  282 
Typholvsin,  219 
Tyramiiie,  584r-586 

Tyrosinase,  61,  66,  67,  68,  112,  473,  477 
Tyrosine,  88,  91,  95,  271,  310,  357,  391, 

472,  500,  550-555,  578,  583,  586-589, 

591,  616,  641 
Tyrotoxicon,  593 

Ultra-violet  light,  215 

rays,  177,  179,  208,  209,  375,  378,  461 
Uncinaria  duodenalis,  136 
Unicellular  organisms,  immunity  in,  246 
Uracil,  104,  627 
Urate  calculi,  461 

deposits,  histology  of,  639 
Urea,  26,  199,  212,  244,  357,  532-540 
Urease,  56,  61,  110 
Uremia,  65,  355,  420,  532-540,  568 
Uric  acid,  311,  357,  466,  468,  494,  510, 
542,  623,  626-641 

calculi,  460 

concretions,  310 

infarcts,  640 
Uricase,  66,  90 
Urinary  calculi,  459-464 
Urobilin,   296,  303,  306,  467,  481,  483, 

494,  495 
Urobilinogen,  481 
Urochrome,  461 
Uroerythrin,  461 
Uro-fuscin,  485 
Uroleucic  acid,  587 
Urorosein,  583 
Urostealith  calculi,  463 
Urticaria,  197,  343,  351,  352,  586 
factitia,  382 


IJschinsky's  medium,  10.3 
I'terine  autolysis,  Sti 
Uterus,  inyolutioii  of,  39«i 

Valkuianw  acid,  l.M 
Vegetable  hemolysins,  220  223 

poi.sons,  21S 
Venom,  21.S,  320,  325 

cobra,  223,  224 

enzymes  in,  14  \ 

hemolysis  by,  223  224 

platypus.  l.")0 

snake,  141   150,  2 IS 
Viperida>,  142 

Viscosity  of  blood,  293,  .'515 
Vital  stains,  45 
Vitamines,  104,  280  289,  378,  513 

fat  .soluble,  462 
Vomiting,  cyclic,  .56S 

pernicious,  of  pregnancy,  546 

War  dropsy,  285 

Wasps,  154 

Wassermann  reaction,  163,  229  233 

Waste  pigments,  478 

Water  absorbing  capacity  of  colloids, 

336 
Waxy  degeneration,  197,  394 

of  muscles,  396 
Wound  repair,  277 

secretions,  362 

Xanthelasma,  419,  480 
Xanthine,  627 

bodies,  627 

calculi,  463 

o.\ida.se,  66 
Xanthochromia,  362 
Xanthoma,  493 

tuberosum  multiplex,  518 
Xanthophyll,  479 
Xanthosis  diabetica,  480 
Xerophthalmia,  284 
X-rays,  70,  98,  99,   120,   122,  209,  31  J, 

376-378,  506 
Xylose,  502,  506 

Yeasts,  162,  246 

Zein,  280 
Zooprecipitins,  187 
Zooto.xins,  141-158 
Zymogen,  55,  84 
Zymoids,  57,  t>2 
Zymophore,  209 
Zymoplastic  substance,  316 


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'■;!ii(DlosFv»' 

liihr-niry 

J  6  194  f 

OCT  9     1941 

MOV  1  ?  114 

1 

JUN  1  r    ]QAA 

^  ID    '^^^ 

1944 

LD  21-100m-7,'39(402.s) 

THE  UNIVERSITY  OF  CALIFORNIA  UBRARY 


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